Recombinant polypeptides and methods of use thereof

ABSTRACT

The present disclosure provides recombinant polypeptides, nucleic acids encoding the recombinant polypeptides and methods for using these polypeptides and/or nucleic acids in enhancing or inducing an immune response in a subject in need thereof. The present disclosure also provides methods of treating a cell proliferative disorder, such as cancer, by administering the disclosed polypeptides and/or nucleic acids to a subject in need thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 21, 2017, isnamed IMHC-001/F01US_ST25.txt and is 42 KB in size.

BACKGROUND OF THE INVENTION

Immunogenic cell death is a form of cell death or apoptosis. Unliketraditional apoptosis, which is mostly non-immunogenic, immunogenic celldeath in cancer cells can induce an effective anti-tumor immune responsethrough the activation of dendritic cells. The pre-apoptotic state isdefined as the state before the activation of Caspase 3/7, themanifestation of cell apoptosis. Immunogenic cell death is characterizedby the expression of pre-apoptotic damage-associated-molecular-patterns(DAMPs) on the surface of a dying cell. There are three importantpre-apoptotic DAMPs which are exposed to the cell surface duringimmunogenic cell death: calreticulin (CRT), HSP70 and HSP90. These threepre-apoptotic DAMP signals play an important role in dendritic cellrecruitment and cell phagocytosis by CRT and dendritic cellmaturation/activation by HSP70 and HSP90, resulting in effectiveanti-tumor immune response. Selected forms of chemotherapy andradiotherapy can induce collateral immunogenic cell death. While thesetherapies can induce one or two of the three pre-apoptotic DAMP signals,they do not induce the expression of all three pre-apoptotic DAMPsignals. Furthermore, chemotherapy and radiotherapy areimmunosuppressive therapies, which reduce numbers of lymphocytes andalso cause collateral damages to surrounding non-tumor cells, resultingin poor anti-tumor immune responses and also adverse eventsrespectively.

There is a need for compositions that induce immunogenic cell death withincreased efficiency and potency by inducing the expression of all threepre-apoptotic DAMP signals, while minimizing adverse effects. Thepresent disclosure addresses this need.

SUMMARY OF THE INVENTION

The present disclosure provides acidic recombinant polypeptides.

The present disclosure provides recombinant polypeptides comprising,consisting essentially of, or consisting of, an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-16 or an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to any of the amino acid sequences of SEQ ID NO:1-16. In one aspect, the recombinant polypeptides comprise, consistessentially of, or consist of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-8 or an amino acid sequence that is atleast about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto any of the amino acid sequences of SEQ ID NO: 1-8. In one aspect, therecombinant polypeptides comprise, consist essentially of, or consist ofan amino acid sequence selected from the group consisting of SEQ ID NO:9-16 or an amino acid sequence that is at least about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98% or about 99% identical to any of the amino acidsequences of SEQ ID NO: 9-16. In a preferred aspect, the recombinantpolypeptide comprises, consists essentially of, or consists of an aminoacid sequence of SEQ ID NO: 9 or amino acid sequence that is at leastabout 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98% or about 99% identical to anyof the amino acid sequences of SEQ ID NO: 9.

In one aspect, the recombinant polypeptides comprise, consistessentially of, or consist of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-16 or amino acid sequence that is atleast about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto any of the amino acid sequences of SEQ ID NO: 1-16 that are acidicrecombinant polypeptides as determined by pI. In one aspect, the pI ofthe recombinant polypeptide is lower than the pI of SEQ ID NO: 1-16. Inone aspect, aspartic acid (D), glutamic acid (E) and leucine (L) areeach independently present in an amount greater than, or equal to, theamount of any other amino acid residue present within the recombinantpolypeptide sequence. In one aspect, the amino acid sequence of theacidic recombinant polypeptides comprise aspartic acid (D), glutamicacid (E) and leucine (L) as the three most abundant residues within theamino acid sequence or as greater than or equal to in abundance to thenext most abundant amino acid residue of the acidic recombinantpolypeptide.

The present disclosure also provides nucleic acid molecules encoding anyof the recombinant polypeptides disclosed herein. In one aspect, thenucleic acid molecules encode the recombinant polypeptides of SEQ ID NO:1-16. In one aspect, the nucleic acid molecule comprises, consistsessentially of, or consists of a nucleic acid sequence of SEQ ID NO:17-32 or a nucleic acid sequence that is at least about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98% or about 99% identical to any of the nucleic acidsequence of SEQ ID NO: 17-32.

The present disclosure also provides expression vectors or plasmidscomprising any of the nucleic acids disclosed herein. The presentdisclosure also provides host cells comprising any of the recombinantpolypeptides and/or nucleic acids disclosed herein. The presentdisclosure also provides pharmaceutical compositions comprising any ofthe recombinant polypeptides and/or nucleic acids disclosed herein, anda pharmaceutically acceptable carrier. In one aspect, thepharmaceutically acceptable carrier comprises a buffer solutioncomprising a NaCl concentration from about 0.4M to about 1.0M at a pH ofabout 7.5 to about 9.0.

The present disclosure provides a method of enhancing or inducing animmune response in a subject in need thereof comprising administering tothe subject any of the recombinant polypeptides and/or nucleic acidsdisclosed herein. The present invention provides a method of enhancingor inducing an immune response in a subject in need thereof comprisingadministering to the subject a pharmaceutical composition comprising anyof the recombinant polypeptides and/or nucleic acids disclosed herein

In one aspect, the immune response is by immunogenic cell death. In oneaspect, the immunogenic cell death comprises endogenous dendritic cellactivation. In one aspect, the cells have increased expression ofpre-apoptotic Damage-Associated-Molecular-Pattern (DAMP) signalscomprising of calreticulin (CRT), Heat Shock Protein 70 (HSP70), HeatShock Protein 90 (HSP90), or a combination thereof. In one aspect, thecell is a cancer cell. In one aspect, the cancer cell is selected from agroup comprising of lung cancer, colon cancer or breast cancer cells.

The present disclosure provides a method of enhancing or inducing theendogenous presentation of disease-associated antigens on a cell surfacein a subject in need thereof, comprising administering to the subjectany of the recombinant polypeptides and/or nucleic acids disclosedherein. The present disclosure provides a method of enhancing orinducing the endogenous presentation of disease-associated antigens on acell surface in a subject in need thereof, comprising administering tothe subject a pharmaceutical composition comprising any of therecombinant polypeptides and/or nucleic acids disclosed herein. In oneaspect, the cell is a cancer cell. In one aspect, the cancer cell isselected from a group comprising of lung cancer, colon cancer or breastcancer cells.

The present disclosure provides a method of treating, preventing oralleviating at least one of the symptoms of a cell proliferativedisorder in a subject in need thereof comprising administering to saidsubject a therapeutically effective amount of any of the recombinantpolypeptides and/or nucleic acids disclosed herein. The presentdisclosure provides a method of treating, preventing or alleviating atleast one of the symptoms of a cell proliferative disorder in a subjectin need thereof comprising administering to said subject atherapeutically effective amount of a pharmaceutical compositioncomprising any of the recombinant polypeptides and/or nucleic acidsdisclosed herein. In one aspect, the cell proliferative disorder is acancer. In one aspect, the cancer is selected from a group comprising oflung cancer, colon cancer or breast cancer.

The present disclosure also provides a kit comprising the compositionsdisclosed herein for performing any of the methods disclosed herein.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

As used herein, the singular forms of a word also include the pluralform of the word, unless the context clearly dictates otherwise; asexamples, the terms “a,” “an,” and “the” are understood to be singularor plural and the term “or” is understood to be inclusive. By way ofexample, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises,” will be understood to imply the inclusion of a statedelement, integer or step, or group of elements, integers or steps, butnot the exclusion of any other element, integer or step, or group ofelements, integers or steps. Throughout the specification the word“consisting of,” or variations such as “consists of,” will be understoodto imply the inclusion of a stated element, integer or step, or group ofelements, integers or steps, and the exclusion of any other element,integer or step, or group of elements, integers or steps. Throughout thespecification the word “consisting essentially of,” or variations suchas “consists essentially of,” will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, and any other element, integer or step, or group of elements,integers or steps that do not materially affect the basic and novelcharacteristics of the claimed invention.

About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwiseclear from the context, all numerical values provided herein aremodified by the term “about.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The references cited hereinare not admitted to be prior art to the claimed disclosure. In the caseof conflict, the present Specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting. Other featuresand advantages of the disclosure will be apparent from the followingdetailed description and claim.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of some characteristics of immunogeniccell death. Tumor cells marked for cell death have cell surfaceexpression of pre-apoptotic Damage-Associated-Molecular-Pattern (DAMP)signals such as calreticulin (CRT), HSP70 and HSP90. Dendritic cells areactivated upon the recognition of DAMP signals. Mature dendritic cellsmigrate to lymph nodes and can in turn prime CD4+ and CD8+ T-cells,which are important for mediating immunogenic cell death.

FIG. 2 shows a graph depicting the molecular weight of CRYA_1Brecombinant polypeptide (SEQ ID NO: 9) of about 20 kDa as determined bymass spectrometry.

FIG. 3A shows a bar graph quantifying the percentage of cells thatexpress CRT(Caltreticulin) following treatment of H441 human lung cancercell lines (HTB-174, ATCC) with various concentrations of CRYA_1Brecombinant polypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinantpolypeptide (SEQ ID NO: 9) was diluted to the final concentration of 35,50, 75 μg/ml and the H441 cells were incubated for 1 hour at 37° C. TheCRT-expressing H441 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using CRT mAb (Abcam). FIG. 3B shows theflow cytometry profiles used for quantification.

FIG. 4A shows a bar graph quantifying the percentage of cells thatexpress CRT following treatment of H460 human lung cancer cell lines(HTB-177, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 0.1, 1, 10, 25,35, 50 μg/ml and the H460 cells were incubated for 30 minutes at 37° C.The CRT-expressing H460 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using CRT mAb (Abcam). FIG. 4B shows theflow cytometry profiles used for quantification.

FIG. 5A shows a bar graph quantifying the percentage of cells thatexpress CRT following treatment of HCT15 human colon cancer cell lines(CCL-225, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the HCT15 cells were incubated for 55 minutes at 37° C. TheCRT-expressing HCT15 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using CRT mAb (Abcam). FIG. 5B shows theflow cytometry profiles used for quantification.

FIG. 6A shows a bar graph quantifying the percentage of cells thatexpress CRT following treatment of MCF7 human breast cancer cell lines(HTB-22, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the MCF7 cells were incubated for 1 hour and 10 minutes at 37°C. The CRT-expressing MCF7 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using CRT mAb (Abcam). FIG. 6B shows theflow cytometry profiles used for quantification.

FIG. 7A shows a bar graph quantifying the percentage of cells thatexpress HSP70 (70 kDa heat shock protein) following treatment of H441human lung cancer cell lines (HTB-174, ATCC) with various concentrationsof CRYA_1B recombinant polypeptide (SEQ ID NO: 9). The stock CRYA_1Brecombinant polypeptide (SEQ ID NO: 9) was diluted to the finalconcentration of 35, 50, 75 μg/ml and the H441 cells were incubated for1 hour and 50 minutes at 37° C. The Hsp70-expressing H441 cells weredetermined by FACSCalibur (BD Biosciences) flow cytometry using Hsp70mAb (Enzo Life Sciences). FIG. 7B shows the flow cytometry profiles usedfor quantification.

FIG. 8A shows a bar graph quantifying the percentage of cells thatexpress HSP70 following treatment of H460 human lung cancer cell lines(HTB-177, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 0.1, 1, 10, 25,35, 50 μg/ml and the H460 cells were incubated for 1 hour and 15 minutesat 37° C. The Hsp70-expressing H460 cells were determined by FACSCalibur(BD Biosciences) flow cytometry using Hsp70 mAb (Enzo Life Sciences).FIG. 8B shows the flow cytometry profiles used for quantification.

FIG. 9A shows a bar graph quantifying the percentage of cells thatexpress HSP70 following treatment of HCT15 human colon cancer cell lines(CCL-225, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the HCT15 cells were incubated for 1 hour and 50 minutes at37° C. The Hsp70-expressing HCT15 cells were determined by FACSCalibur(BD Biosciences) flow cytometry using Hsp70 mAb (Enzo Life Sciences).FIG. 9B shows the flow cytometry profiles used for quantification.

FIG. 10A shows a bar graph quantifying the percentage of cells thatexpress HSP70 following treatment of MCF7 human breast cancer cell lines(HTB-22, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the MCF7 cells were incubated for 1 hour and 40 minutes at 37°C. The Hsp70-expressing MCF7 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using Hsp70 mAb (Enzo Life Sciences). FIG.10B shows the flow cytometry profiles used for quantification.

FIG. 11A shows a bar graph quantifying the percentage of cells thatexpress HSP90 (90 kDa heat shock protein) following treatment of H441human lung cancer cell lines (HTB-174, ATCC) with various concentrationsof CRYA_1B recombinant polypeptide (SEQ ID NO: 9). The stock CRYA_1Brecombinant polypeptide (SEQ ID NO: 9) was diluted to the finalconcentration of 35, 50, 75 μg/ml and the H441 cells were incubated for1 hour and 40 minutes at 37° C. The Hsp90-expressing H441 cells weredetermined by FACSCalibur (BD Biosciences) flow cytometry using Hsp90mAb (Enzo Life Sciences). FIG. 11B shows the flow cytometry profilesused for quantification.

FIG. 12A shows a bar graph quantifying the percentage of cells thatexpress HSP90 following treatment of H460 human lung cancer cell lines(HTB-177, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 0.1, 1, 10, 25,35, 50 μg/ml and the H460 cells were incubated for 1 hour and 5 minutesat 37° C. The Hsp90-expressing H460 cells were determined by FACSCalibur(BD Biosciences) flow cytometry using Hsp90 mAb (Enzo Life Sciences).FIG. 12B shows the flow cytometry profiles used for quantification.

FIG. 13A shows a bar graph quantifying the percentage of cells thatexpress HSP90 following treatment of HCT15 human colon cancer cell lines(CCL-225, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the HCT15 cells were incubated for 1 hour and 30 minutes at37° C. The Hsp90-expressing HCT15 cells were determined by FACSCalibur(BD Biosciences) flow cytometry using Hsp90 mAb (Enzo Life Sciences).FIG. 13B shows the flow cytometry profiles used for quantification.

FIG. 14A shows a bar graph quantifying the percentage of cells thatexpress HSP90 following treatment of MCF7 human breast cancer cell lines(HTB-22, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the MCF7 cells were incubated for 1 hour and 45 minutes at 37°C. The Hsp90-expressing MCF7 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using Hsp90 mAb (Enzo Life Sciences). FIG.14B shows the flow cytometry profiles used for quantification.

FIG. 15A shows a bar graph quantifying the percentage of cells thatexpress Caspase 3/7 following treatment of H441 human lung cancer celllines (HTB-174, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50 and 75μg/ml and the H441 cells were incubated for 2 hours and 45 minutes at37° C. The H441 cells were determined by FACSCalibur (BD Biosciences)flow cytometry using Caspase 3/7 (Invitrogen) assay. FIG. 15B shows theflow cytometry profiles used for quantification.

FIG. 16A shows a bar graph quantifying the percentage of cells thatexpress Caspase 3/7 following treatment of H460 human lung cancer celllines (HTB-177, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 0.1, 1, 10, 25,35 and 50 μg/ml and the H460 cells were incubated for 2 hours and 45minutes at 37° C. The H460 cells were determined by FACSCalibur (BDBiosciences) flow cytometry using Caspase 3/7 (Invitrogen) assay. FIG.16B shows the flow cytometry profiles used for quantification.

FIG. 17A shows a bar graph quantifying the percentage of cells thatexpress Caspase 3/7 following treatment of HCT15 human colon cancer celllines (CCL-225, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the HCT15 cells were incubated for 2 hours and 30 minutes at37° C. The HCT15 cells were determined by FACSCalibur (BD Biosciences)flow cytometry using Caspase 3/7 (Invitrogen) assay. FIG. 17B shows theflow cytometry profiles used for quantification.

FIG. 18A shows a bar graph quantifying the percentage of cells thatexpress Caspase 3/7 following treatment of MCF7 human breast cancer celllines (HTB-22, ATCC) with various concentrations of CRYA_1B recombinantpolypeptide (SEQ ID NO: 9). The stock CRYA_1B recombinant polypeptide(SEQ ID NO: 9) was diluted to the final concentration of 35, 50, 75μg/ml and the MCF7 cells were incubated for 2 hours and 45 minutes at37° C. The MCF7 cells were determined by FACSCalibur (BD Biosciences)flow cytometry using Caspase 3/7 (Invitrogen) assay. FIG. 18B shows theflow cytometry profiles used for quantification.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides recombinant polypeptides, and thenucleic acids encoding these polypeptides, pharmaceutical compositionscomprising these polypeptides and/or nucleic acids, and methods forusing these polypeptides and/or nucleic acids to enhance or induce animmune response in a subject in need thereof

Compositions of the Present Disclosure

The present disclosure provides recombinant polypeptides that comprise,consist essentially of, or consist of, any of the amino acid sequencesshown in Table 1A. The present disclosure also provides recombinantpolypeptides comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to any of the amino acid sequencesshown in Table 1A.

The present disclosure also provides acidic recombinant polypeptidevariants that comprise, consist essentially of, or consist of, any ofthe amino acid sequences shown in Table 1A, wherein the recombinantpolypeptide variant is acidic as determined by isoelectric point (pI).The present disclosure also provides acidic recombinant polypeptidevariants comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to any of the amino acid sequencesshown in Table 1A.

An “acidic variant” is a variant of a polypeptide of interest which ismore acidic (e.g., as determined by calculation of pI) than the parentor original polypeptide of interest. The “pI” or “isoelectric point” ofa polypeptide refers to the pH at which the polypeptide's positivecharge balances its negative charge. pI can be calculated by any meansknown in the art, for example, from the net charge of the amino acidresidues of the polypeptide or can be determined by isoelectricfocusing.

In some aspects, an acidic variant is derived from the original parentsequence by making amino acid substitutions. A first mutationalsubstitution is made by substituting any basic amino acid (K, R or H),neutral non-polar amino acid (G, A, V, L, I, M, F, W or P) or neutralpolar amino acid (S, T, C, Y, N or Q) of the original parent sequencewith an acidic amino acid (D or E). A second mutational substitution ismade by making the inverse mutational substitution of the firstmutational substitution. For example, all serine (S) residues fromoriginal parent sequence are substituted with glutamic acid (E) residues(first substitution). In addition, all glutamic acid (E) residues fromthe original parent sequence are substituted with serine (S) residues(second substitution). In one aspect, the inverse substitutionscomprise, consist essentially of or consist of the mutation of allserine (S) residues of the original parent sequence to glutamic acid (E)and the mutation of all glutamic acid (E) residues of original parentsequence to serine (S) residues; the mutation of all serine (S) residuesof the original parent sequence to aspartic acid (D) and the mutation ofall aspartic acid (D) residues of the original parent sequence to serine(S) residues; the mutation of all valine (V) residues of the originalparent sequence to aspartic acid (D) and the mutation of all asparticacid (D) of the original parent sequence to valine (V) residues; or themutation of all serine (S) residues of the original parent sequence toleucine (L) residues and the mutation of all leucine (L) residues of theoriginal parent sequence to serine (S) residues. In a preferred aspect,the amino acid substitutions result in a recombinant polypeptide whereaspartic acid (D), glutamic acid (E) and leucine (L) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within the recombinantpolypeptide sequence. In a preferred aspect, the amino acidsubstitutions result in a recombinant polypeptide with leucine (L),aspartic acid (D) and glutamic acid (E) as the three most abundant aminoacid residues of the acidic variant or as greater than or equal to inabundance to the next most abundant amino acid residue of the acidicvariant. In some aspects, multiple inverse mutational substitutions ofamino acids can be made.

The present disclosure also provides acidic recombinant polypeptidevariants that comprise, consist essentially of, or consist of, any ofthe amino acid sequences shown in Table 1A, wherein the recombinantpolypeptide variant is acidic as determined by isoelectric point (pI),wherein the pI of the recombinant peptide variant is lower than the pIof peptide sequence from which the recombinant peptide was derived, andwherein leucine (L), aspartic acid (D) and glutamic acid (E) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within the recombinantpolypeptide sequence. The present disclosure also provides acidicrecombinant polypeptide variants that comprise, consist essentially of,or consist of, any of the amino acid sequences shown in Table 1A,wherein the recombinant polypeptide variant is acidic as determined byisoelectric point (pI) and wherein leucine (L), aspartic acid (D) andglutamic acid (E) are the three most abundant amino acid residues of theacidic variant or are greater than or equal to in abundance to the nextmost abundant amino acid residue of the acidic variant. The presentdisclosure also provides acidic recombinant polypeptide variantscomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to any of the amino acid sequences shown in Table1A.

TABLE 1A Recombinant Polypeptide Sequences Name Amino Acid SequenceSEQ ID NO: Anser cygnoides domesticus MDITIQHPTA7FKRALGPLIPS 1 CRYAARLFDQFFGEGLLEYDLLPLF SSTISPYYRQSLFRSVLESG ISEVRSDRDKFTIMLDVKHFSPEDLSVKIIDDFVEIHGKH SERQDDHGYISREFHRRYRL PANVDQSAITCSLSGDGMLTFSGPKVPSNMDPTHSERPIP VSREEKPTSAPSS Rhea americana MDITIQHPWFKRALGPLIPS 2CRYAA RLFDQFFGEGLLEYDLLPLF SSTISPYYRQSLFRSVLESG ISEVRSDREKFTIMLDVKHFSPEDLSVKIIDDFVEIHGKH SERQDDHGYISREFHRRYRL PSNVDQSAITCSLSSDGMLTFSGPKVQANMDPSHSERPIP VSREEKPTSAPSS Anas platyrhynchosRALGPLIPSRLFDQFFGEGL 3 CRYAA LEYDLLPLFSSTISPYYRQS LFRSVLESGISEVRSDRDKFTIMLDVKHFSPEDLSVKIID DFVEIHGKHSERQDDHGYIS REFHRRYRLPANVDQSAITCSLSGDGMLTFSGPKVPSNMD PTHSERPIP Anas platyrhynchos MDITIHNPLIRRPLFSWLAP 4CRYAB SRIFDQIFGEHLQESELLPA SPSLSPFLMRSPIFRMPSWL ETGLSEMRLEKDKFSVNLDVKHFSPEELKVKVLGDMVEIH GKHEERQDEHGFIAREFNRK YRIPADVDPLTITSSLSLDGVLTVSAPRKQSDVPERSIPI TREEKPAIAGAQRK Homo sapiens MDVTIQHPTWFKRTLGPFYPS 5CRYAA RLFDQFFGEGLFEYDLLPFL SSTISPYYRQSLFRTVLDSG ISEVRSDRDKFVIFLDVKHFSPEDLTVKVQDDFVEIHGKH NERQDDHGYISREFHRRYRL PSNVDQSALSCSLSADGMLTFCGPKIQTGLDATHAERAIP VSREEKPTSAPSS Drosophila melanogasterMANIPLLLSLADDLGRMSMV 6 HSP23 PFYEPYYCQRQRNPYLALVG PMEQQLRQLEKQVGASSGSSGAVSKIGKDGFQVCMDVSHF KPSELVVKVQDNSVLVEGNH EEREDDHGFITRHFVRRYALPPGYEADKVASTLSSDGVLT IKVPKPPAIEDKGNERIVQI QQVGPAHLNVKENPKEAVEQ DNGNDKDrosophila melanogaster MRSLPMFWRMAEEMARMPRL 7 H5P22SSPFHAFFHEPPVWSVALPR NWQHIARWQEQELAPPATVN KDGYKLTLDVKDYSELKVKVLDESVVLVEAKSEQQEAEQG GYSSRHFLGRYVLPDGYEAD KVSSSLSDDGVLTISVPNPPGVQETLKEREVTIEQTGEPA KKSAEEPKDKTASQ Anser cygnoides domesticusMDITIHNPLIRRPLFSWLAP 8 CRYAB SRIFDQIFGEHLQESELLPA SPSLSPFLMRSPIFRMPSWLETGLSEMRLEKDKFSVNLDV KHFSPEELKVKVLGDMVEIH GKHEERQDEHGFIAREFNRKYRIPADVDPLTITSSLSLDG VLTVSAPRKQSDVPERSIPI TREEKPAIAGAQRK

In a preferred aspect, the present disclosure provides recombinantpolypeptides that comprise, consist essentially of, or consist of, anyof the amino acid sequences shown in Table 1B. The present disclosurealso provides recombinant polypeptides that have an amino acid sequencethat is at least about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to any of the amino acid sequences shown in Table 1B.

TABLE 1B Recombinant Polypeptide Sequences Name Amino Acid SequenceSEQ ID NO: CRYA_1B MDITIQHPWFKRALGPLIPE 9 Anser cygnoides domesticusRLFDQFFGSGLLSYDLLPLF CRYAA EETIEPYYRQELFREVLSEG IESVREDRDKFTIMLDVKHFEPSDLEVKIIDDFVSIHGKH ESRQDDHGYIERSFHRRYRL PANVDQEAITCELEGDGMLTFEGPKVPENMDPTHESRPIP VERSSKPTEAPEE Rhea americana MDITIQHPWFKRALGPLIPE10 CRYAA RLFDQFFGSGLLSYDLLPLF EETIEPYYRQELFREVLSEG IESVREDRSKFTIMLDVKHFEPSDLEVKIIDDFVSIHGKH ESRQDDHGYIERSFHRRYRL PENVDQEAITCELEEDGMLTFEGPKVQANMDPEHESRPIP VERSSKPTEAPEE Anas platyrhynchosRALGPLIPERLFDQFFGSGL 11 CRYAA LSYDLLPLFEETIEPYYRQE LFREVLSEGIESVREDRDKFTIMLDVKHFEPSDLEVKIID DFVSIHGKHESRQDDHGYIE RSFHRRYRLPANVDQEAITCELEGDGMLTFEGPKVPENMD PTHESRPIP Anas platyrhynchos MSITIHNPLIRRPLFDWLAP12 CRYAB DRIFSQIFGEHLQEDELLPA DPDLDPFLMRDPIFRMPDWL ETGLDEMRLEKSKFDVNLSVKHFDPEELKVKVLGSMVEIH GKHEERQSEHGFIAREFNRK YRIPASVSPLTITDDLDLSGVLTVDAPRKQDSVPERDIPI TREEKPAIAGAQRK Homo sapiens MDVTIQHPWFKRTLGPFYPE 13CRYAA RLFDQFFGSGLFSYDLLPFL EETIEPYYRQELFRTVLDEG IESVREDRDKFVIFLDVKHFEPSDLTVKVQDDFVSIHGKH NSRQDDHGYIERSFHRRYRL PENVDQEALECELEADGMLTFCGPKIQTGLDATHASRAIP VERSSKPTEAPEE Drosophila melanogasterMANIPLLLSLAVVLGRMSMD 14 HSP23 PFYEPYYCQRQRNPYLALDG PMEQQLRQLEKQDGASSGSSGADSKIGKVGFQDCMVDSHF KPSELDDKDQVNSDLDEGNH EEREVVHGFITRHFDRRYALPPGYEAVKDASTLSSVGDLT IKDPKPPAIEVKGNERIDQI QQDGPAHLNDKENPKEADEQ VNGNVKDrosophila melanogaster MRLSPMFWRMAEEMARMPRS 15 HSP22LLPFHAFFHEPPDWLDASPR NWQHIARWQEQESAPPATDN KVGYKSTSVDKVYLESKDKDSVELDDSDEAKLEQQEAEQG GYLLRHFSGRYDSPVGYEAV KDLLLSLVVGDSTILDPNPPGDQETSKEREDTIEQTGEPA KKLAEEPKVKTALQ Anser cygnoides domesticusMSITIHNPLIRRPLFDWLAP 16 CRYAB DRIFSQIFGEHLQEDELLPA DPDLDPFLMRDPIFRMPDWLETGLDEMRLEKSKFDVNLSV KHFDPEELKVKVLGSMVEIH GKHEERQSEHGFIAREFNRKYRIPASVSPLTITDDLDLSG VLTVDAPRKQDSVPERDIPI TREEKPAIAGAQRK

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Anser cygnoidesdomesticus alpha-A-crystallin (CRYAA) (GenBank # XP_013036875.1)comprising, consisting essentially of, or consisting of, the amino acidsequence of SEQ ID NO: 1 or a recombinant polypeptide comprising,consisting essentially of, or consisting of, an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the amino acid sequence of SEQ ID NO: 1.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from Ansercygnoides domesticus alpha-A-crystallin (CRYAA) (GenBank #XP_013036875.1) comprising, consisting essentially of, or consisting of,the amino acid sequence of SEQ ID NO: 1, wherein the alpha crystallinrecombinant polypeptide variant is acidic as determined by isoelectricpoint (pI), or an acidic alpha crystallin recombinant polypeptidevariant comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 1, wherein the alpha crystallin recombinant polypeptide variant isacidic as determined by isoelectric point (pI). In some aspects, the pIof the recombinant polypeptide is lower than the pI of SEQ ID NO: 1. Insome aspects, leucine (L), aspartic acid (D) and glutamic acid (E) areeach independently present in an amount greater than, or equal to, theamount of any other amino acid residue present within the recombinantpolypeptide sequence. In some aspects, leucine (L), aspartic acid (D)and glutamic acid (E) are the three most abundant amino acid residues inthe acidic alpha crystallin recombinant polypeptide variant or aregreater than or equal to in abundance to the next most abundant aminoacid residue of the acidic alpha crystallin recombinant polypeptidevariant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Anser cygnoides domesticus alpha-A-crystallin (CRYAA)(GenBank # XP_013036875.1) comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 9 or a recombinantpolypeptide comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 9. For example, SEQ ID NO:9 has at least 80% sequence identity tothe polypeptide of SEQ ID NO:1, SEQ ID NO:9 is acidic as determined bypI, the pI of SEQ ID NO:9 is lower than the pI of SEQ ID NO:1, andaspartic acid (D), glutamic acid (E) and leucine (L) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within SED ID NO:9 (that is,glutamic acid (E) is 23 residues, leucine (L) is 15 residues, andaspartic acid (D) is 14 residues of the 173 amino acid sequence of SEQID NO:9, with proline (P) (14 residues) being the next most presentamino acid residue within SEQ ID NO:9).

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Rhea Americanaalpha-A-crystallin (CRYAA) (GenBank # P02505.1) comprising, consistingessentially of, or consisting of, the amino acid sequence of SEQ ID NO:2 or a recombinant polypeptide comprising, consisting essentially of, orconsisting of, an amino acid sequence that is at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% identical to the amino acidsequence of SEQ ID NO: 2.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from RheaAmericana alpha-A-crystallin (CRYAA) (GenBank # P02505.1) comprising,consisting essentially of, or consisting of, the amino acid sequence ofSEQ ID NO: 2, wherein the alpha crystallin recombinant polypeptidevariant is acidic as determined by isoelectric point (pI), or an acidicalpha crystallin recombinant polypeptide variant comprising, consistingessentially of, or consisting of, an amino acid sequence that is atleast about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto the amino acid sequence of SEQ ID NO: 2, wherein the alpha crystallinrecombinant polypeptide variant is acidic as determined by isoelectricpoint (pI). In some aspects, the pI of the recombinant polypeptide islower than the pI of SEQ ID NO: 2. In some aspects, leucine (L),aspartic acid (D) and glutamic acid (E) are each independently presentin an amount greater than, or equal to, the amount of any other aminoacid residue present within the recombinant polypeptide sequence. Insome aspects, leucine (L), aspartic acid (D) and glutamic acid (E) arethe three most abundant amino acid residues in the acidic alphacrystallin recombinant polypeptide variant or are greater than or equalto in abundance to the next most abundant amino acid residue of theacidic alpha crystallin recombinant polypeptide variant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Rhea Americana alpha-A-crystallin (CRYAA) (GenBank #P02505.1) comprising, consisting essentially of, or consisting of, theamino acid sequence of SEQ ID NO: 10 or a recombinant polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to the amino acid sequence of SEQ ID NO: 10. Forexample, SEQ ID NO:10 has at least 75% sequence identity to thepolypeptide of SEQ ID NO:2, SEQ ID NO:10 is acidic as determined by pI,the pI of SEQ ID NO:10 is lower than the pI of SEQ ID NO:2, and asparticacid (D), glutamic acid (E) and leucine (L) are each independentlypresent in an amount greater than, or equal to, the amount of any otheramino acid residue present within SED ID NO:10.

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Anasplatyrhynchos alpha-A-crystallin (CRYAA) (GenBank #O12984.1) comprising,consisting essentially of, consisting of, the amino acid sequence of SEQID NO: 3 or a recombinant polypeptide comprising, consisting essentiallyof, consisting of, an amino acid sequence that is at least about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98% or about 99% identical to the amino acidsequence of SEQ ID NO: 3.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from Anasplatyrhynchos alpha-A-crystallin (CRYAA) (GenBank #O12984.1) comprising,consisting essentially of, or consisting of, the amino acid sequence ofSEQ ID NO: 3, wherein the alpha crystallin recombinant polypeptidevariant is acidic as determined by isoelectric point (pI), or an acidicalpha crystallin recombinant polypeptide variant comprising, consistingessentially of, or consisting of, an amino acid sequence that is atleast about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto the amino acid sequence of SEQ ID NO: 3, wherein the alpha crystallinrecombinant polypeptide variant is acidic as determined by isoelectricpoint (pI). In some aspects, the pI of the recombinant polypeptide islower than the pI of SEQ ID NO: 3. In some aspects, leucine (L),aspartic acid (D) and glutamic acid (E) are each independently presentin an amount greater than, or equal to, the amount of any other aminoacid residue present within the recombinant polypeptide sequence. Insome aspects, leucine (L), aspartic acid (D) and glutamic acid (E) arethe three most abundant amino acid residues in the acidic alphacrystallin recombinant polypeptide variant or are greater than or equalto in abundance to the next most abundant amino acid residue of theacidic alpha crystallin recombinant polypeptide variant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Anas platyrhynchos alpha-A-crystallin (CRYAA) (GenBank#O12984.1) comprising, consisting essentially of, or consisting of, theamino acid sequence of SEQ ID NO: 11 or a recombinant polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to the amino acid sequence of SEQ ID NO: 11. Forexample, SEQ ID NO:11 has at least 80% sequence identity to thepolypeptide of SEQ ID NO:3, SEQ ID NO:11 is acidic as determined by pI,the pI of SEQ ID NO:11 is lower than the pI of SEQ ID NO:3, and asparticacid (D), glutamic acid (E) and leucine (L) are each independentlypresent in an amount greater than, or equal to, the amount of any otheramino acid residue present within SED ID NO:11.

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Anasplatyrhynchos alpha-B-crystallin (CRYAB) (GenBank # Q05557.1)comprising, consisting essentially of, consisting of, the amino acidsequence of SEQ ID NO: 4 or a recombinant polypeptide comprising,consisting essentially of, consisting of, an amino acid sequence that isat least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the amino acid sequence of SEQ ID NO: 4.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from Anasplatyrhynchos alpha-B-crystallin (CRYAB) (GenBank # Q05557.1)comprising, consisting essentially of, or consisting of, the amino acidsequence of SEQ ID NO: 4, wherein the alpha crystallin recombinantpolypeptide variant is acidic as determined by isoelectric point (pI),or an acidic alpha crystallin recombinant polypeptide variantcomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to the amino acid sequence of SEQ ID NO: 4, whereinthe alpha crystallin recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI). In some aspects, the pI of therecombinant polypeptide is lower than the pI of SEQ ID NO: 4. In someaspects, leucine (L), aspartic acid (D) and glutamic acid (E) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within the recombinantpolypeptide sequence. In some aspects, leucine (L), aspartic acid (D)and glutamic acid (E) are the three most abundant amino acid residues inthe acidic alpha crystallin recombinant polypeptide variant or aregreater than or equal to in abundance to the next most abundant aminoacid residue of the acidic alpha crystallin recombinant polypeptidevariant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Anas platyrhynchos alpha-B-crystallin (CRYAB) (GenBank #Q05557.1) comprising, consisting essentially of, or consisting of, theamino acid sequence of SEQ ID NO: 12 or a recombinant polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to the amino acid sequence of SEQ ID NO: 12. Forexample, SEQ ID NO:12 has at least 80% sequence identity to thepolypeptide of SEQ ID NO:4, SEQ ID NO:12 is acidic as determined by pI,the pI of SEQ ID NO:12 is lower than the pI of SEQ ID NO:4, and asparticacid (D), glutamic acid (E) and leucine (L) are each independentlypresent in an amount greater than, or equal to, the amount of any otheramino acid residue present within SED ID NO:12.

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Homo sapiensalpha-A-crystallin (CRYAA) (GenBank # AAH69528.1) comprising, consistingessentially of, or consisting of, the amino acid sequence of SEQ ID NO:5 or a recombinant polypeptide comprising, consisting essentially of, orconsisting of, an amino acid sequence that is at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% identical to the amino acidsequence of SEQ ID NO: 5.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from Homosapiens alpha-A-crystallin (CRYAA) (GenBank # AAH69528.1) comprising,consisting essentially of, or consisting of, the amino acid sequence ofSEQ ID NO: 5, wherein the alpha crystallin recombinant polypeptidevariant is acidic as determined by isoelectric point (pI), or an acidicalpha crystallin recombinant polypeptide variant comprising, consistingessentially of, or consisting of, an amino acid sequence that is atleast about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98% or about 99% identicalto the amino acid sequence of SEQ ID NO: 5, wherein the alpha crystallinrecombinant polypeptide variant is acidic as determined by isoelectricpoint (pI). In some aspects, the pI of the recombinant polypeptide islower than the pI of SEQ ID NO: 5. In some aspects, leucine (L),aspartic acid (D) and glutamic acid (E) are each independently presentin an amount greater than, or equal to, the amount of any other aminoacid residue present within the recombinant polypeptide sequence. Insome aspects, leucine (L), aspartic acid (D) and glutamic acid (E) arethe three most abundant amino acid residues in the acidic alphacrystallin recombinant polypeptide variant or are greater than or equalto in abundance to the next most abundant amino acid residue of theacidic alpha crystallin recombinant polypeptide variant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Homo sapiens alpha-A-crystallin (CRYAA) (GenBank #AAH69528.1) comprising, consisting essentially of, or consisting of, theamino acid sequence of SEQ ID NO: 13 or a recombinant polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to the amino acid sequence of SEQ ID NO: 13. Forexample, SEQ ID NO:13 has at least 80% sequence identity to thepolypeptide of SEQ ID NO:5, SEQ ID NO:13 is acidic as determined by pI,the pI of SEQ ID NO:13 is lower than the pI of SEQ ID NO:5, and asparticacid (D), glutamic acid (E) and leucine (L) are each independentlypresent in an amount greater than, or equal to, the amount of any otheramino acid residue present within SED ID NO:13.

The present disclosure provides an HSP23 recombinant polypeptidesequence or amino acid sequence derived from Drosophila melanogasterHSP23 (GenBank # AAA28637.1) comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 6 or a recombinantpolypeptide comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 6.

The present disclosure provides an HSP23 recombinant polypeptide variantsequence or amino acid sequence derived from Drosophila melanogasterHSP23 (GenBank # AAA28637.1) comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 6, wherein theHSP23 recombinant polypeptide variant is acidic as determined byisoelectric point (pI), or an acidic HSP23 recombinant polypeptidevariant comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 6, wherein the HSP23 recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI). In some aspects, the pI of therecombinant polypeptide is lower than the pI of SEQ ID NO: 6. In someaspects, leucine (L), aspartic acid (D) and glutamic acid (E) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within the recombinantpolypeptide sequence. In some aspects, leucine (L), aspartic acid (D)and glutamic acid (E) are the three most abundant amino acid residues inthe acidic HSP23 recombinant polypeptide variant or are greater than orequal to in abundance to the next most abundant amino acid residue ofthe acidic HSP23 recombinant polypeptide variant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Drosophila melanogaster HSP23 (GenBank # AAA28637.1)comprising, consisting essentially of, or consisting of, the amino acidsequence of SEQ ID NO: 14 or a recombinant polypeptide comprising,consisting essentially of, or consisting of, an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the amino acid sequence of SEQ ID NO: 14. For example, SEQID NO:14 has at least 80% sequence identity to the polypeptide of SEQ IDNO:6, SEQ ID NO:14 is acidic as determined by pI, the pI of SEQ ID NO:14is lower than the pI of SEQ ID NO:6, and aspartic acid (D), glutamicacid (E) and leucine (L) are each independently present in an amountgreater than, or equal to, the amount of any other amino acid residuepresent within SED ID NO:14.

The present disclosure provides an HSP22 recombinant polypeptidesequence or amino acid sequence derived from Drosophila melanogasterHSP22 (GenBank # AAA28635.1) comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 7 or a recombinantpolypeptide having an amino acid sequence that is at least about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98% or about 99% identical to the amino acidsequence of SEQ ID NO: 7.

The present disclosure provides an acidic HSP22 recombinant polypeptidevariant sequence or amino acid sequence derived from Drosophilamelanogaster HSP22 (GenBank # AAA28635.1) comprising, consistingessentially of, or consisting of, the amino acid sequence of SEQ ID NO:7, wherein the HSP22 recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI), or an acidic HSP22 recombinantpolypeptide variant comprising, consisting essentially of, or consistingof, an amino acid sequence that is at least about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98% or about 99% identical to the amino acid sequence of SEQID NO: 7, wherein the HSP22 recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI). In some aspects, the pI of therecombinant polypeptide is lower than the pI of SEQ ID NO: 7. In someaspects, leucine (L), aspartic acid (D) and glutamic acid (E) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within the recombinantpolypeptide sequence. In some aspects, leucine (L), aspartic acid (D)and glutamic acid (E) are the three most abundant amino acid residues inthe acidic HSP22 recombinant polypeptide variant or are greater than orequal to in abundance to the next most abundant amino acid residue ofthe acidic HSP22 recombinant polypeptide variant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Drosophila melanogaster HSP22 (GenBank # AAA28635.1)comprising, consisting essentially of, or consisting of, the amino acidsequence of SEQ ID NO: 15 or a recombinant polypeptide comprising,consisting essentially of, or consisting of, an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the amino acid sequence of SEQ ID NO: 15. For example, SEQID NO:15 has at least 65% sequence identity to the polypeptide of SEQ IDNO:7, SEQ ID NO:15 is acidic as determined by pI, the pI of SEQ ID NO:15is lower than the pI of SEQ ID NO:7, and aspartic acid (D), glutamicacid (E) and leucine (L) are each independently present in an amountgreater than, or equal to, the amount of any other amino acid residuepresent within SED ID NO:15.

The present disclosure provides an alpha crystallin recombinantpolypeptide sequence or amino acid sequence derived from Anser cygnoidesdomesticus alpha-B-crystallin (CRYAB) (GenBank # XP_013042703.1)comprising, consisting essentially of, or consisting of, the amino acidsequence of SEQ ID NO: 8 or a recombinant polypeptide comprising,consisting essentially of, or consisting of, an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to the amino acid sequence of SEQ ID NO: 8.

The present disclosure provides an acidic alpha crystallin recombinantpolypeptide variant sequence or amino acid sequence derived from Ansercygnoides domesticus alpha-B-crystallin (CRYAB) (GenBank #XP_013042703.1) comprising, consisting essentially of, or consisting of,the amino acid sequence of SEQ ID NO: 8, wherein the alpha crystallinrecombinant polypeptide variant is acidic as determined by isoelectricpoint (pI), or an acidic alpha crystallin recombinant polypeptidevariant comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 8, wherein the alpha crystallin recombinant polypeptide variant isacidic as determined by isoelectric point (pI). In some aspects, the pIof the recombinant polypeptide is lower than the pI of SEQ ID NO: 8. Insome aspects, leucine (L), aspartic acid (D) and glutamic acid (E) areeach independently present in an amount greater than, or equal to, theamount of any other amino acid residue present within the recombinantpolypeptide sequence. In some aspects, leucine (L), aspartic acid (D)and glutamic acid (E) are the three most abundant amino acid residues inthe acidic alpha crystallin recombinant polypeptide variant or aregreater than or equal to in abundance to the next most abundant aminoacid residue of the acidic alpha crystallin recombinant polypeptidevariant.

In a preferred aspect, the recombinant polypeptide is an acidic variantderived from Anser cygnoides domesticus alpha-B-crystallin (CRYAB)(GenBank # XP_013042703.1) comprising, consisting essentially of, orconsisting of, the amino acid sequence of SEQ ID NO: 16 or a recombinantpolypeptide comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of SEQ IDNO: 16. For example, SEQ ID NO:16 has at least 80% sequence identity tothe polypeptide of SEQ ID NO:8, SEQ ID NO:16 is acidic as determined bypI, the pI of SEQ ID NO:16 is lower than the pI of SEQ ID NO:8, andaspartic acid (D), glutamic acid (E) and leucine (L) are eachindependently present in an amount greater than, or equal to, the amountof any other amino acid residue present within SED ID NO:16.

The present disclosure provides an isolated nucleic acid moleculeencoding a recombinant polypeptide that comprises, consists essentiallyof, or consists of, any of the amino acid sequences shown in Table 1A.The present disclosure also provides isolated nucleic acid moleculesencoding recombinant polypeptides comprising, consisting essentially of,or consisting of, an amino acid sequence that is at least about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98% or about 99% identical to any of theamino acid sequences shown in Table 1A.

The present disclosure also provides an isolated nucleic acid moleculeencoding a recombinant polypeptide variant that comprises, consistsessentially of, or consists of, any of the amino acid sequences shown inTable 1A, wherein the recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI). The present disclosure alsoprovides isolated nucleic acid molecules encoding a recombinantpolypeptide variants comprising, consisting essentially of, orconsisting of, an amino acid sequence that is at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% identical to any of the aminoacid sequences shown in Table 1A.

The present disclosure also provides isolated nucleic acid moleculesencoding acidic recombinant polypeptide variants that comprise, consistessentially of, or consist of, any of the amino acid sequences shown inTable 1A, wherein the recombinant polypeptide variant is acidic asdetermined by isoelectric point (pI), wherein the pI of the recombinantpeptide variant is lower than the pI of peptide sequence from which therecombinant peptide was derived, and wherein leucine (L), aspartic acid(D) and glutamic acid (E) are each independently present in an amountgreater than, or equal to, the amount of any other amino acid residuepresent within the recombinant polypeptide sequence. The presentdisclosure also provides isolated nucleic acid molecules encoding acidicrecombinant polypeptide variants that comprise, consist essentially of,or consist of, any of the amino acid sequences shown in Table 1A,wherein the recombinant polypeptide variant is acidic as determined byisoelectric point (pI) and wherein leucine (L), aspartic acid (D) andglutamic acid (E) are the three most abundant amino acid sequences ofthe acidic variant or are greater than or equal to in abundance to thenext most abundant amino acid residue of the acidic alpha crystallinrecombinant polypeptide variant. The present disclosure also providesisolated nucleic acid molecules encoding acid recombinant polypeptidevariants comprising, consisting essentially of, or consisting of, anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to any of the amino acid sequencesshown in Table 1A.

The present disclosure also provides isolated nucleic acid moleculesthat comprise, consist essentially of, or consist of, any of the nucleicacid sequences shown in Table 2A. The present disclosure also providesnucleic acid molecules comprising, consisting essentially of, orconsisting of, a nucleic acid sequence that is at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% identical to any of the nucleicacid sequences shown in Table 2A.

TABLE 2A Nucleic Acid Sequences Name Nucleic Acid Sequence SEQ ID NO:Anser cygnoides domesticus ATGGATATTACCATTCAGCA 17 CRYAATCCGTGGTTTAAACGCGCGC TGGGCCCGCTGATTCCGAGC CGCCTGTTTGATCAGTTTTTTGGCGAAGGCCTGCTGGAAT ATGATCTGCTGCCGCTGTTT AGCAGCACCATTAGCCCGTATTATCGCCAGAGCCTGTTTC GCAGCGTGCTGGAAAGCGGC ATTAGCGAAGTGCGCAGCGATCGCGATAAATTTACCATTA TGCTGGATGTGAAACATTTT AGCCCGGAAGATCTGAGCGTGAAAATTATTGATGATTTTG TGGAAATTCATGGCAAACAT AGCGAACGCCAGGATGATCATGGCTATATTAGCCGCGAAT TTCATCGCCGCTATCGCCTG CCGGCGAACGTGGATCAGAGCGCGATTACCTGCAGCCTGA GCGGCGATGGCATGCTGACC TTTAGCGGCCCGAAAGTGCCGAGCAACATGGATCCGACCC ATAGCGAACGCCCGATTCCG GTGAGCCGCGAAGAAAAACCGACCAGCGCGCCGAGCAGC Rhea americana ATGGATATTACCATTCAGCA 18 CRYAATCCGTGGTTTAAACGCGCGC TGGGCCCGCTGATTCCGAGC CGCCTGTTTGATCAGTTTTTTGGCGAAGGCCTGCTGGAAT ATGATCTGCTGCCGCTGTTT AGCAGCACCATTAGCCCGTATTATCGCCAGAGCCTGTTTC GCAGCGTGCTGGAAAGCGGC ATTAGCGAAGTGCGCAGCGATCGCGAAAAATTTACCATTA TGCTGGATGTGAAACATTTT AGCCCGGAAGATCTGAGCGTGAAAATTATTGATGATTTTG TGGAAATTCATGGCAAACAT AGCGAACGCCAGGATGATCATGGCTATATTAGCCGCGAAT TTCATCGCCGCTATCGCCTG CCGAGCAACGTGGATCAGAGCGCGATTACCTGCAGCCTGA GCAGCGATGGCATGCTGACC TTTAGCGGCCCGAAAGTGCAGGCGAACATGGATCCGAGCC ATAGCGAACGCCCGATTCCG GTGAGCCGCGAAGAAAAACCGACCAGCGCGCCGAGCAGC Anas platyrhynchos CGCGCGCTGGGCCCGCTGAT 19 CRYAATCCGAGCCGCCTGTTTGATC AGTTTTTTGGCGAAGGCCTG CTGGAATATGATCTGCTGCCGCTGTTTAGCAGCACCATTA GCCCGTATTATCGCCAGAGC CTGTTTCGCAGCGTGCTGGAAAGCGGCATTAGCGAAGTGC GCAGCGATCGCGATAAATTT ACCATTATGCTGGATGTGAAACATTTTAGCCCGGAAGATC TGAGCGTGAAAATTATTGAT GATTTTGTGGAAATTCATGGCAAACATAGCGAACGCCAGG ATGATCATGGCTATATTAGC CGCGAATTTCATCGCCGCTATCGCCTGCCGGCGAACGTGG ATCAGAGCGCGATTACCTGC AGCCTGAGCGGCGATGGCATGCTGACCTTTAGCGGCCCGA AAGTGCCGAGCAACATGGAT CCGACCCATAGCGAACGCCC GATTCCGAnas platyrhynchos ATGGATATTACCATTCATAA 20 CRYAB CCCGCTGATTCGCCGCCCGCTGTTTAGCTGGCTGGCGCCG AGCCGCATTTTTGATCAGAT TTTTGGCGAACATCTGCAGGAAAGCGAACTGCTGCCGGCG AGCCCGAGCCTGAGCCCGTT TCTGATGCGCAGCCCGATTTTTCGCATGCCGAGCTGGCTG GAAACCGGCCTGAGCGAAAT GCGCCTGGAAAAAGATAAATTTAGCGTGAACCTGGATGTG AAACATTTTAGCCCGGAAGA ACTGAAAGTGAAAGTGCTGGGCGATATGGTGGAAATTCAT GGCAAACATGAAGAACGCCA GGATGAACATGGCTTTATTGCGCGCGAATTTAACCGCAAA TATCGCATTCCGGCGGATGT GGATCCGCTGACCATTACCAGCAGCCTGAGCCTGGATGGC GTGCTGACCGTGAGCGCGCC GCGCAAACAGAGCGATGTGCCGGAACGCAGCATTCCGATT ACCCGCGAAGAAAAACCGGC GATTGCGGGCGCGCAGCGCA AAHomo sapiens ATGGATGTGACCATTCAGCA 21 CRYAA TCCGTGGTTTAAACGCACCCTGGGCCCGTTTTATCCGAGC CGCCTGTTTGATCAGTTTTT TGGCGAAGGCCTGTTTGAATATGATCTGCTGCCGTTTCTG AGCAGCACCATTAGCCCGTA TTATCGCCAGAGCCTGTTTCGCACCGTGCTGGATAGCGGC ATTAGCGAAGTGCGCAGCGA TCGCGATAAATTTGTGATTTTTCTGGATGTGAAACATTTT AGCCCGGAAGATCTGACCGT GAAAGTGCAGGATGATTTTGTGGAAATTCATGGCAAACAT AACGAACGCCAGGATGATCA TGGCTATATTAGCCGCGAATTTCATCGCCGCTATCGCCTG CCGAGCAACGTGGATCAGAG CGCGCTGAGCTGCAGCCTGAGCGCGGATGGCATGCTGACC TTTTGCGGCCCGAAAATTCA GACCGGCCTGGATGCGACCCATGCGGAACGCGCGATTCCG GTGAGCCGCGAAGAAAAACC GACCAGCGCGCCGAGCAGCDrosophila melanogaster ATGGCGAACATTCCGCTGCT 22 HSP23GCTGAGCCTGGCGGATGATC TGGGCCGCATGAGCATGGTG CCGTTTTATGAACCGTATTATTGCCAGCGCCAGCGCAACC CGTATCTGGCGCTGGTGGGC CCGATGGAACAGCAGCTGCGCCAGCTGGAAAAACAGGTGG GCGCGAGCAGCGGCAGCAGC GGCGCGGTGAGCAAAATTGGCAAAGATGGCTTTCAGGTGT GCATGGATGTGAGCCATTTT AAACCGAGCGAACTGGTGGTGAAAGTGCAGGATAACAGCG TGCTGGTGGAAGGCAACCAT GAAGAACGCGAAGATGATCATGGCTTTATTACCCGCCATT TTGTGCGCCGCTATGCGCTG CCGCCGGGCTATGAAGCGGATAAAGTGGCGAGCACCCTGA GCAGCGATGGCGTGCTGACC ATTAAAGTGCCGAAACCGCCGGCGATTGAAGATAAAGGCA ACGAACGCATTGTGCAGATT CAGCAGGTGGGCCCGGCGCATCTGAACGTGAAAGAAAACC CGAAAGAAGCGGTGGAACAG GATAACGGCAACGATAAADrosophila melanogaster ATGCGCAGCCTGCCGATGTT 23 HSP22TTGGCGCATGGCGGAAGAAA TGGCGCGCATGCCGCGCCTG AGCAGCCCGTTTCATGCGTTTTTTCATGAACCGCCGGTGT GGAGCGTGGCGCTGCCGCGC AACTGGCAGCATATTGCGCGCTGGCAGGAACAGGAACTGG CGCCGCCGGCGACCGTGAAC AAAGATGGCTATAAACTGACCCTGGATGTGAAAGATTATA GCGAACTGAAAGTGAAAGTG CTGGATGAAAGCGTGGTGCTGGTGGAAGCGAAAAGCGAAC AGCAGGAAGCGGAACAGGGC GGCTATAGCAGCCGCCATTTTCTGGGCCGCTATGTGCTGC CGGATGGCTATGAAGCGGAT AAAGTGAGCAGCAGCCTGAGCGATGATGGCGTGCTGACCA TTAGCGTGCCGAACCCGCCG GGCGTGCAGGAAACCCTGAAAGAACGCGAAGTGACCATTG AACAGACCGGCGAACCGGCG AAAAAAAGCGCGGAAGAACCGAAAGATAAAACCGCGAGCCAG Anser cygnoides domesticus ATGGATATTACCATTCATAA24 CRYAB CCCGCTGATTCGCCGCCCGC TGTTTAGCTGGCTGGCGCCG AGCCGCATTTTTGATCAGATTTTTGGCGAACATCTGCAGG AAAGCGAACTGCTGCCGGCG AGCCCGAGCCTGAGCCCGTTTCTGATGCGCAGCCCGATTT TTCGCATGCCGAGCTGGCTG GAAACCGGCCTGAGCGAAATGCGCCTGGAAAAAGATAAAT TTAGCGTGAACCTGGATGTG AAACATTTTAGCCCGGAAGAACTGAAAGTGAAAGTGCTGG GCGATATGGTGGAAATTCAT GGCAAACATGAAGAACGCCAGGATGAACATGGCTTTATTG CGCGCGAATTTAACCGCAAA TATCGCATTCCGGCGGATGTGGATCCGCTGACCATTACCA GCAGCCTGAGCCTGGATGGC GTGCTGACCGTGAGCGCGCCGCGCAAACAGAGCGATGTGC CGGAACGCAGCATTCCGATT ACCCGCGAAGAAAAACCGGCGATTGCGGGCGCGCAGCGCA AA

In a preferred aspect, the present disclosure provides isolated nucleicacid molecules that comprise, consist essentially of, or consist of, anyof the nucleic acid sequences shown in Table 2B. The present disclosurealso provides nucleic acid molecules comprising, consisting essentiallyof, or consisting of, a nucleic acid sequence that is at least about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98% or about 99% identical to any ofthe nucleic acid sequences shown in Table 2B.

TABLE 2B Nucleic Acid Sequences Name Nucleic Acid Sequence SEQ ID NO:CRYA_1B ATGGATATTACCATTCAGCA 25 Anser cygnoides domesticusTCCGTGGTTTAAACGCGCGC CRYAA TGGGCCCGCTGATTCCGGAA CGCCTGTTTGATCAGTTTTTTGGCAGCGGCCTGCTGAGCT ATGATCTGCTGCCGCTGTTT GAAGAAACCATTGAACCGTATTATCGCCAGGAACTGTTTC GCGAAGTGCTGAGCGAAGGC ATTGAAAGCGTGCGCGAAGATCGCGATAAATTTACCATTA TGCTGGATGTGAAACATTTT GAACCGAGCGATCTGGAAGTGAAAATTATTGATGATTTTG TGAGCATTCATGGCAAACAT GAAAGCCGCCAGGATGATCATGGCTATATTGAACGCAGCT TTCATCGCCGCTATCGCCTG CCGGCGAACGTGGATCAGGAAGCGATTACCTGCGAACTGG AAGGCGATGGCATGCTGACC TTTGAAGGCCCGAAAGTGCCGGAAAACATGGATCCGACCC ATGAAAGCCGCCCGATTCCG GTGGAACGCAGCAGCAAACCGACCGAAGCGCCGGAAGAA Rhea americana ATGGATATTACCATTCAGCA 26 CRYAATCCGTGGTTTAAACGCGCGC TGGGCCCGCTGATTCCGGAA CGCCTGTTTGATCAGTTTTTTGGCAGCGGCCTGCTGAGCT ATGATCTGCTGCCGCTGTTT GAAGAAACCATTGAACCGTATTATCGCCAGGAACTGTTTC GCGAAGTGCTGAGCGAAGGC ATTGAAAGCGTGCGCGAAGATCGCAGCAAATTTACCATTA TGCTGGATGTGAAACATTTT GAACCGAGCGATCTGGAAGTGAAAATTATTGATGATTTTG TGAGCATTCATGGCAAACAT GAAAGCCGCCAGGATGATCATGGCTATATTGAACGCAGCT TTCATCGCCGCTATCGCCTG CCGGAAAACGTGGATCAGGAAGCGATTACCTGCGAACTGG AAGAAGATGGCATGCTGACC TTTGAAGGCCCGAAAGTGCAGGCGAACATGGATCCGGAAC ATGAAAGCCGCCCGATTCCG GTGGAACGCAGCAGCAAACCGACCGAAGCGCCGGAAGAA Anas platyrhynchos CGCGCGCTGGGCCCGCTGAT 27 CRYAATCCGGAACGCCTGTTTGATC AGTTTTTTGGCAGCGGCCTG CTGAGCTATGATCTGCTGCCGCTGTTTGAAGAAACCATTG AACCGTATTATCGCCAGGAA CTGTTTCGCGAAGTGCTGAGCGAAGGCATTGAAAGCGTGC GCGAAGATCGCGATAAATTT ACCATTATGCTGGATGTGAAACATTTTGAACCGAGCGATC TGGAAGTGAAAATTATTGAT GATTTTGTGAGCATTCATGGCAAACATGAAAGCCGCCAGG ATGATCATGGCTATATTGAA CGCAGCTTTCATCGCCGCTATCGCCTGCCGGCGAACGTGG ATCAGGAAGCGATTACCTGC GAACTGGAAGGCGATGGCATGCTGACCTTTGAAGGCCCGA AAGTGCCGGAAAACATGGAT CCGACCCATGAAAGCCGCCC GATTCCGAnas platyrhynchos ATGAGCATTACCATTCATAA 28 CRYAB CCCGCTGATTCGCCGCCCGCTGTTTGATTGGCTGGCGCCG GATCGCATTTTTAGCCAGAT TTTTGGCGAACATCTGCAGGAAGATGAACTGCTGCCGGCG GATCCGGATCTGGATCCGTT TCTGATGCGCGATCCGATTTTTCGCATGCCGGATTGGCTG GAAACCGGCCTGGATGAAAT GCGCCTGGAAAAAAGCAAATTTGATGTGAACCTGAGCGTG AAACATTTTGATCCGGAAGA ACTGAAAGTGAAAGTGCTGGGCAGCATGGTGGAAATTCAT GGCAAACATGAAGAACGCCA GAGCGAACATGGCTTTATTGCGCGCGAATTTAACCGCAAA TATCGCATTCCGGCGAGCGT GAGCCCGCTGACCATTACCGATGATCTGGATCTGAGCGGC GTGCTGACCGTGGATGCGCC GCGCAAACAGGATAGCGTGCCGGAACGCGATATTCCGATT ACCCGCGAAGAAAAACCGGC GATTGCGGGCGCGCAGCGCA AAHomo sapiens ATGGATGTGACCATTCAGCA 29 CRYAA TCCGTGGTTTAAACGCACCCTGGGCCCGTTTTATCCGGAA CGCCTGTTTGATCAGTTTTT TGGCAGCGGCCTGTTTAGCTATGATCTGCTGCCGTTTCTG GAAGAAACCATTGAACCGTA TTATCGCCAGGAACTGTTTCGCACCGTGCTGGATGAAGGC ATTGAAAGCGTGCGCGAAGA TCGCGATAAATTTGTGATTTTTCTGGATGTGAAACATTTT GAACCGAGCGATCTGACCGT GAAAGTGCAGGATGATTTTGTGAGCATTCATGGCAAACAT AACAGCCGCCAGGATGATCA TGGCTATATTGAACGCAGCTTTCATCGCCGCTATCGCCTG CCGGAAAACGTGGATCAGGA AGCGCTGGAATGCGAACTGGAAGCGGATGGCATGCTGACC TTTTGCGGCCCGAAAATTCA GACCGGCCTGGATGCGACCCATGCGAGCCGCGCGATTCCG GTGGAACGCAGCAGCAAACC GACCGAAGCGCCGGAAGAADrosophila melanogaster ATGGCGAACATTCCGCTGCT 30 HSP23GCTGAGCCTGGCGGTGGTGC TGGGCCGCATGAGCATGGAT CCGTTTTATGAACCGTATTATTGCCAGCGCCAGCGCAACC CGTATCTGGCGCTGGATGGC CCGATGGAACAGCAGCTGCGCCAGCTGGAAAAACAGGATG GCGCGAGCAGCGGCAGCAGC GGCGCGGATAGCAAAATTGGCAAAGTGGGCTTTCAGGATT GCATGGTGGATAGCCATTTT AAACCGAGCGAACTGGATGATAAAGATCAGGTGAACAGCG ATCTGGATGAAGGCAACCAT GAAGAACGCGAAGTGGTGCATGGCTTTATTACCCGCCATT TTGATCGCCGCTATGCGCTG CCGCCGGGCTATGAAGCGGTGAAAGATGCGAGCACCCTGA GCAGCGTGGGCGATCTGACC ATTAAAGATCCGAAACCGCCGGCGATTGAAGTGAAAGGCA ACGAACGCATTGATCAGATT CAGCAGGATGGCCCGGCGCATCTGAACGATAAAGAAAACC CGAAAGAAGCGGATGAACAG GTGAACGGCAACGTGAAADrosophila melanogaster ATGCGCCTGAGCCCGATGTT 31 HSP22TTGGCGCATGGCGGAAGAAA TGGCGCGCATGCCGCGCAGC CTGCTGCCGTTTCATGCGTTTTTTCATGAACCGCCGGATT GGCTGGATGCGAGCCCGCGC AACTGGCAGCATATTGCGCGCTGGCAGGAACAGGAAAGCG CGCCGCCGGCGACCGATAAC AAAGTGGGCTATAAAAGCACCAGCGTGGATAAAGTGTATC TGGAAAGCAAAGATAAAGAT AGCGTGGAACTGGATGATAGCGATGAAGCGAAACTGGAAC AGCAGGAAGCGGAACAGGGC GGCTATCTGCTGCGCCATTTTAGCGGCCGCTATGATAGCC CGGTGGGCTATGAAGCGGTG AAAGATCTGCTGCTGAGCCTGGTGGTGGGCGATAGCACCA TTCTGGATCCGAACCCGCCG GGCGATCAGGAAACCAGCAAAGAACGCGAAGATACCATTG AACAGACCGGCGAACCGGCG AAAAAACTGGCGGAAGAACCGAAAGTGAAAACCGCGCTGCAG Anser cygnoides domesticus ATGAGCATTACCATTCATAA32 CRYAB CCCGCTGATTCGCCGCCCGC TGTTTGATTGGCTGGCGCCG GATCGCATTTTTAGCCAGATTTTTGGCGAACATCTGCAGG AAGATGAACTGCTGCCGGCG GATCCGGATCTGGATCCGTTTCTGATGCGCGATCCGATTT TTCGCATGCCGGATTGGCTG GAAACCGGCCTGGATGAAATGCGCCTGGAAAAAAGCAAAT TTGATGTGAACCTGAGCGTG AAACATTTTGATCCGGAAGAACTGAAAGTGAAAGTGCTGG GCAGCATGGTGGAAATTCAT GGCAAACATGAAGAACGCCAGAGCGAACATGGCTTTATTG CGCGCGAATTTAACCGCAAA TATCGCATTCCGGCGAGCGTGAGCCCGCTGACCATTACCG ATGATCTGGATCTGAGCGGC GTGCTGACCGTGGATGCGCCGCGCAAACAGGATAGCGTGC CGGAACGCGATATTCCGATT ACCCGCGAAGAAAAACCGGCGATTGCGGGCGCGCAGCGCAAA

The present disclosure also provides pharmaceutical compositionscomprising the recombinant polypeptides or nucleic acids disclosedherein.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

In one aspect, the pharmaceutical composition can comprise, consistessentially of, or consist of any one of the recombinant polypeptidesdisclosed herein in a pharmaceutically acceptable carrier. In someaspects, the pharmaceutical composition is formulated as an aqueousformulation. The aqueous formulation can comprise, consist essentiallyof, or consist of a salt buffer that may be selected from, but is notlimited to, NaCl, KCl, and NaOAc. In a preferred aspect, the salt buffercomprises NaCl. In a more preferred aspect, the NaCl is at aconcentration from about 0.4M to about 1.0M. In one aspect, the pH ofthe buffer solution is between about 7.5 and about 9.0.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for the treatment ofcancer, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities, taken orally orapplied through the skin with patches.

The term “therapeutically effective amount,” as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In one aspect, thedisease or condition to be treated is a cell proliferative disorder. Ina preferred aspect, the disease or condition to be treated is cancer.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freezedrying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterilefiltered solutionthereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

The pharmaceutical compositions can include co-formulations of any ofthe recombinant polypeptides and nucleic acids described herein.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The present disclosure also provides plasmids, expression vectors andhost cells comprising the recombinant polypeptides disclosed herein andthe nucleic acid molecules encoding the recombinant polypeptidesdisclosed herein. In one aspect, the disclosure provides a plasmid or anexpression vector comprising a nucleic acid molecule, the moleculecomprising a nucleotide sequence of any one of SEQ ID NO: 17-32, or anucleic acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to any of the nucleic acid sequence ofSEQ ID NO: 17-32, or a fragment thereof. In one aspect, the disclosureprovides a host cell comprising a recombinant polypeptide comprising anamino acid sequence of any one of SEQ ID NO: 1-16, or an amino acidsequence that is at least about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% identical to any of the amino acid sequences of SEQ ID NO:1-16, or a fragment thereof, or a host cell comprising a nucleic acidmolecule comprising a nucleic acid sequence of any one of SEQ ID NO:17-32, or a nucleic acid sequence that is at least about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98% or about 99% identical to any of the nucleic acidsequence of SEQ ID NO: 17-32, or a fragment thereof.

As used herein, the term “transformation,” “transfection,” and“transduction” refer to the transfer of nucleic acid (i.e., a nucleotidepolymer) into a cell. As used herein, the term “genetic transformation”refers to the transfer and incorporation of DNA, especially recombinantDNA, into a cell. The transferred nucleic acid can be introduced into acell via an expression vector.

Polynucleotide molecules comprising a desired polynucleotide sequenceare propagated by placing the molecule in a vector. Viral and non-viralvectors can be used, including plasmids. The choice of plasmid willdepend on the type of cell in which propagation is desired and thepurpose of propagation. Certain vectors are useful for amplifying andmaking large amounts of the desired DNA sequence. Other vectors aresuitable for expression in cells in culture. Still other vectors aresuitable for transfer and expression in cells in a whole animal orperson. The choice of appropriate vector is well within the skill of theart. Many such vectors are available commercially. The partial orfull-length polynucleotide is inserted into a vector typically by meansof DNA ligase attachment to a cleaved restriction enzyme site in thevector. Alternatively, the desired nucleotide sequence can be insertedby homologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Toexpress a nucleic acid encoding a polypeptide disclosed herein, anucleic acid molecule encoding the polypeptide, operably linked toregulatory sequences that control transcriptional expression in anexpression vector, is introduced into a host cell. In addition totranscriptional regulatory sequences, such as promoters and enhancers,expression vectors can include translational regulatory sequences and amarker gene which is suitable for selection of cells that carry theexpression vector. The gene product encoded by a polynucleotide of thedisclosure is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.In the expression vector, the polypeptide-encoding polynucleotide islinked to a regulatory sequence as appropriate to obtain the desiredexpression properties. These can include promoters, enhancers,terminators, operators, repressors, and inducers. The promoters can beregulated (e.g., the promoter from the steroid inducible pIND vector(Invitrogen)) or constitutive (e.g., promoters from CMV, SV40,Elongation Factor, or LTR sequences). These are linked to the desirednucleotide sequence using the techniques described above for linkage tovectors. Any techniques known in the art can be used. Accordingly, theexpression vector will generally provide a transcriptional andtranslational initiation region, which can be inducible or constitutive,where the coding region is operably linked under the transcriptionalcontrol of the transcriptional initiation region, and a transcriptionaland translational termination region.

An expression cassette (“expression unit”) can be introduced into avariety of vectors, e.g., plasmid, BAC, YAC, bacteriophage such aslambda, P1, M13, etc., plant or animal viral vectors (e.g.,retroviral-based vectors, adenovirus vectors), and the like, where thevectors are normally characterized by the ability to provide selectionof cells comprising the expression vectors. The vectors can provide forextrachromosomal maintenance, particularly as plasmids or viruses, orfor integration into the host chromosome. Where extrachromosomalmaintenance is desired, an origin sequence is provided for thereplication of the plasmid, which can be low or high copy-number. A widevariety of markers are available for selection, particularly those whichprotect against toxins, more particularly against antibiotics. Theparticular marker that is chosen is selected in accordance with thenature of the host, where, in some cases, complementation can beemployed with auxotrophic hosts. Introduction of the DNA construct canuse any convenient method, including, e.g., conjugation, bacterialtransformation, calcium-precipitated DNA, electroporation, fusion,transfection, infection with viral vectors, biolistics, and the like.

Accordingly, polypeptides for use within the present disclosure can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells(including cultured cells of multicellular organisms), particularlycultured mammalian cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook and Russell, Molecular Cloning: A LaboratoryManual (3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001), and Ausubel et al., Short Protocols in MolecularBiology (4th ed., John Wiley & Sons, 1999). For example, the recombinantpolypeptides of the disclosure can be expressed from bacterialEscherichia coli cells.

To direct a recombinant polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence) canbe provided in the expression vector. The secretory signal sequence canbe that of the native form of the recombinant protein, or can be derivedfrom another secreted protein or synthesized de novo. The secretorysignal sequence is operably linked to the polypeptide-encoding DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences can be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Cultured mammalian cells can be suitable hosts for production ofrecombinant polypeptides for use within the present disclosure. Methodsfor introducing exogenous DNA into mammalian host cells include calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978;Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Vander Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al.,supra), and liposome-mediated transfection (Hawley-Nelson et al., Focus15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed by,for example, Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al.,U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Examples of suitable mammalian hostcells include African green monkey kidney cells (Vero; ATCC CRL 1587),human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamsterkidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), caninekidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1;ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, Utah); see also, e.g.,Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitarycells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells(H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1;ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. Strong transcription promoters can be used, such aspromoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No.4,956,288. Other suitable promoters include those from metallothioneingenes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus majorlate promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants.” Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.”Exemplary selectable markers include a gene encoding resistance to theantibiotic neomycin, which allows selection to be carried out in thepresence of a neomycin-type drug, such as G-418 or the like; the gptgene for xanthine-guanine phosphoribosyl transferase, which permits hostcell growth in the presence of mycophenolic acid/xanthine; and markersthat provide resistance to zeocin, bleomycin, blastocidin, andhygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342, 1987;Drocourt et al., Nucl. Acids Res. 18:4009, 1990). Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WO94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See King and Possee, The Baculovirus Expression System: A LaboratoryGuide (Chapman & Hall, London); O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual (Oxford University Press., New York 1994);and Baculovirus Expression Protocols. Methods in Molecular Biology(Richardson ed., Humana Press, Totowa, N.J., 1995). Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, Md.). Thetransfer vector (e.g., PFASTBAC1; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfervectors can include an in-frame fusion with DNA encoding a polypeptideextension or affinity tag as disclosed above. Using techniques known inthe art, a transfer vector containing a protein-encoding DNA sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses the protein or interest is subsequently produced. Recombinantviral stocks are made by methods commonly used in the art.

For protein production, a recombinant virus can be used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVEcells; Invitrogen, Carlsbad, Calif.). See generally Glick and Pasternak,Molecular Biotechnology, Principles & Applications of Recombinant DNA(ASM Press, Washington, D.C., 1994). See also U.S. Pat. No. 5,300,435.Serum-free media are used to grow and maintain the cells. Suitable mediaformulations are known in the art and can be obtained from commercialsuppliers. The cells are grown up from an inoculation density ofapproximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, at which timea recombinant viral stock is added at a multiplicity of infection (MOI)of 0.1 to 10, more typically near 3. Procedures used are generallydescribed in available laboratory manuals (see, e.g., King and Possee,supra; O'Reilly et al., supra; Richardson, supra).

Fungal cells, including yeast cells, can also be used within the presentdisclosure. Yeast species of in this regard include, e.g., Saccharomycescerevisiae, Pichia pastoris, and Pichia methanolica. Methods fortransforming S. cerevisiae cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). An exemplary vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S.Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;5,139,936; and 4,661,454. Transformation systems for other yeasts,including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichiamethanolica, Pichia guillermondii, and Candida maltosa are known in theart. See, e.g., Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986;Cregg, U.S. Pat. No. 4,882,279; and Raymond et al., Yeast 14:11-23,1998. Aspergillus cells can be utilized according to the methods ofMcKnight et al., U.S. Pat. No. 4,935,349. Methods for transformingAcremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No.5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteinsin Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808;5,736,383; 5,854,039; and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus, and other genera are also useful host cells within thepresent disclosure. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well-known in theart (see, e.g., Sambrook and Russell, supra). When expressing arecombinant protein in bacteria such as E. coli, the protein can beretained in the cytoplasm, typically as insoluble granules, or can bedirected to the periplasmic space by a bacterial secretion sequence. Inthe former case, the cells are lysed, and the granules are recovered anddenatured using, for example, guanidine isothiocyanate or urea. Thedenatured protein can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the alternative, the protein canbe recovered from the cytoplasm in soluble form and isolated without theuse of denaturants. The protein is recovered from the cell as an aqueousextract in, for example, phosphate buffered saline. To capture theprotein of interest, the extract is applied directly to achromatographic medium, such as an immobilized antibody orheparin-Sepharose column. Secreted proteins can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media can alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

The recombinant polypeptides can be purified by conventional proteinpurification methods, typically by a combination of chromatographictechniques. See generally Affinity Chromatography: Principles & Methods(Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, ProteinPurification: Principles and Practice (Springer-Verlag, New York 1994).Additional purification steps, such as gel filtration, can be used toobtain the desired level of purity or to provide for desalting, bufferexchange, and the like.

Methods of the Present Disclosure

The present disclosure provides methods for enhancing or inducing animmune response in a subject in need thereof. The subject in needthereof can be a subject with a cell proliferation disorder. In oneaspect, the subject has cancer and the cell is a cancer cell. In apreferred aspect, the subject has lung cancer, colon cancer or breastcancer. In a preferred aspect, the cancer cells can be lung cancercells, colon cancer cells or breast cancer cells.

In one aspect, the methods for enhancing or inducing an immune responsein a subject in need thereof comprise administering at least onerecombinant polypeptide of the present disclosure, or a nucleic acidencoding a recombinant polypeptide of the present disclosure. In oneaspect, the at least one recombinant polypeptide of the presentdisclosure comprises a recombinant polypeptide of SEQ ID NO: 1-8 or anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 9′7%,about 98% or about 99% identical to any of the amino acid sequences ofSEQ ID NO: 1-8 or an acidic variant thereof as described herein. In oneaspect, the at least one recombinant polypeptide of the presentdisclosure comprises a recombinant polypeptide of SEQ ID NO: 9-16 or anamino acid sequence that is at least about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 9′7%,about 98% or about 99% identical to any of the amino acid sequences ofSEQ ID NO: 9-16. In a preferred aspect, the at least one recombinantpolypeptide of the present disclosure comprises a recombinantpolypeptide of SEQ ID NO: 9.

The present disclosure also provides methods for enhancing or inducingthe endogenous presentation of disease associated antigens on a cellsurface in a subject in need thereof. The subject in need thereof can bea subject with a cell proliferation disorder. In one aspect, the subjecthas cancer and the cell is a cancer cell. In a preferred aspect, thesubject has lung cancer, colon cancer or breast cancer. In a preferredaspect, the cancer cells can be lung cancer cells, colon cancer cells orbreast cancer cells.

In one aspect, the methods for enhancing or inducing the endogenouspresentation of disease associated antigens on a cell surface in asubject in need thereof comprise administering at least one recombinantpolypeptide of the present disclosure, or a nucleic acid encoding arecombinant polypeptide of the present disclosure. In one aspect, the atleast one recombinant polypeptide of the present disclosure comprises arecombinant polypeptide of SEQ ID NO: 1-8 or an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to any of the amino acid sequences of SEQ ID NO: 1-8 or anacidic variant thereof as described herein. In one aspect, the at leastone recombinant polypeptide of the present disclosure comprises arecombinant polypeptide of SEQ ID NO: 9-16 or an amino acid sequencethat is at least about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 9′7%, about 98% or about 99%identical to any of the amino acid sequences of SEQ ID NO: 9-16. In apreferred aspect, the at least one recombinant polypeptide of thepresent disclosure comprises a recombinant polypeptide of SEQ ID NO: 9.

In one aspect, enhancing or inducing an “immune response” can be, forexample, a cytokine release response or a humoral (antigen-specific)immune response. The immune response to be enhanced for example, can bean innate immune response, a local immune response, a mucosal immuneresponse or a systemic immune response. As used herein, the terms“enhance” or “enhancing” refer to strengthening (augmenting) of anexisting immune response. The term “inducing” refers to the initiationof an immune response.

In one aspect, “immune response” refers to “immunogenic cell death” or“immunogenic apoptosis”, which is characterized by a robust immuneresponse against antigens expressed by dying cells (FIG. 1). Dyingcells, such as cancer cells, can have an increased expression ofpre-apoptotic Damage-Associated-Molecular-Pattern (DAMP) signalscomprising calreticulin (CRT), HSP70, HSP90, or a combination thereof.In a preferred aspect, the cells have increased expression of each ofCRT, HSP70 and HSP90. Techniques known to one skilled in the art can beused to assess the expression of these cell surface markers. Forexample, the expression of the cell surface markers can be assessedusing standard techniques such as flow cytometry, immunocytochemistry(e.g., staining with tissue specific or cell-marker specificantibodies), fluorescence activated cell sorting (FACS), magneticactivated cell sorting (MACS) or other similar methods known in the art.Fluorescence activated cell sorting (FACS) is a well-known method forseparating particles, including cells, based on the fluorescentproperties of the particles (Kamarch, 1987, Methods Enzymol,151:150-165). Laser excitation of fluorescent moieties in the individualparticles results in a small electrical charge allowing electromagneticseparation of positive and negative particles from a mixture. In oneaspect, cell surface marker-specific antibodies or ligands are labeledwith distinct fluorescent labels. Cells are processed through the flowcytometer, allowing separation of cells based on their ability to bindto the antibodies used. In one aspect, the method of the presentdisclosure induces the expression of pre-apoptotic HSP70, HSP90 orcalreticulin on a cell surface, such as a cancer cell surface.

In one aspect, “immunogenic cell death” or “immunogenic apoptosis”involves the interaction of dendritic cells with a cell, such as acancer cell, leading to a more rapid rate of endogenous dendritic cellactivation, dendritic cell maturation and phagocytosis. The recognitionof pre-apoptotic DAMP signals comprising calreticulin (CRT), HSP70,HSP90, or a combination thereof, by the dendritic cells triggers“endogenous dendritic cell activation”. This leads to “dendritic cellmaturation”, which comprises a redistribution of majorhistocompatibility complex (MHC) molecules from intracellular endocyticcompartments to the dendritic cell surface, down-regulation of antigeninternalization, an increase of surface expression of co-stimulatorymolecules (including CD80 and CD86), cytoskeleton re-organization,secretion of chemokines, cytokines and proteases, surface expression ofadhesion molecules and surface expression of chemokine receptors. Maturedendritic cells that have been exposed to cancer cells dying byimmunogenic cell death can migrate to lymph nodes and induce highnumbers of tumor-specific T lymphocytes (including CD4+ and CD8+ Tcells). This triggers a targeted T-cell mediated response towards thecancer cell. The process of “immunogenic cell death” or “immunogenicapoptosis” is shown in FIG. 1. A person skilled in the art willappreciate that not all techniques known to induce cell death willnecessarily induce immunogenic cell death. Only agents inducingimmunogenic cell death will elicit efficient endogenous dendritic cellactivation. In one aspect an “immune response” refers to endogenousdendritic cell activation, dendritic cell maturation or T-cell mediatedresponse or a combination thereof.

In one aspect, “apoptosis” is the term used to describe the cellsignaling cascade known as programmed cell death. Various therapeuticindications exist for molecules that induce apoptosis (e.g. cancer).Apoptosis can be monitored by any of a number of available techniquesknown and available in the art including, for example, assays thatmeasure fragmentation of DNA, alterations in membrane asymmetry,activation of apoptotic caspases and/or release of cytochrome C and AIF.In one aspect, apoptosis is measured by the activation and expression ofCaspase 3/7.

The present disclosure also provides methods for treating, preventing oralleviating at least one symptom of a cell proliferative disorder in asubject in need thereof. In one aspect, the method is alleviating atleast one symptom of a cell proliferative disorder in a subject in needthereof. In one aspect the cell proliferative disorder is cancer. In apreferred aspect, the cancer is lung cancer, colon cancer or breastcancer.

In one aspect, the methods for treating, preventing or alleviating atleast one symptom of a cell proliferative disorder in a subject in needthereof comprise administering at least one recombinant polypeptide ofthe present disclosure, or a nucleic acid encoding a recombinantpolypeptide of the present disclosure. In one aspect, the at least onerecombinant polypeptide of the present disclosure comprises arecombinant polypeptide of SEQ ID NO: 1-8 or an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to any of the amino acid sequences of SEQ ID NO: 1-8 or anacidic variant thereof as described herein. In one aspect, the at leastone recombinant polypeptide of the present disclosure comprises arecombinant polypeptide of SEQ ID NO: 9-16. In a preferred aspect, theat least one recombinant polypeptide of the present disclosure comprisesa recombinant polypeptide of SEQ ID NO: 9 or an amino acid sequence thatis at least about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98% or about 99%identical to any of the amino acid sequences of SEQ ID NO: 9-16.

As used herein, a “subject” can be any mammal, e.g., a human, a primate,mouse, rat, dog, cat, cow, horse, pig, sheep, goat, camel. In apreferred aspect, the subject is a human. In one aspect, a “subject inneed thereof” is a subject having a cell proliferative disorder, or asubject having an increased risk of developing a cell proliferativedisorder relative to the population at large. In one aspect, a subjectin need thereof has a precancerous condition. In a preferred aspect, asubject in need thereof has cancer.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes decreasing or alleviating the symptoms or complications, oreliminating the disease, condition or disorder. As used herein,“preventing” describes stopping the onset of the symptoms orcomplications of the disease, condition or disorder. As used herein,“alleviating” describes reducing the symptoms or complications ofdisease, condition or disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe disclosure encompass a variety of conditions wherein cell divisionis deregulated. Exemplary cell proliferative disorder include, but arenot limited to, neoplasms, benign tumors, malignant tumors,pre-cancerous conditions, in situ tumors, encapsulated tumors,metastatic tumors, liquid tumors, solid tumors, immunological tumors,hematological tumors, cancers, carcinomas, leukemias, lymphomas,sarcomas, and rapidly dividing cells. The term “rapidly dividing cell”as used herein is defined as any cell that divides at a rate thatexceeds or is greater than what is expected or observed amongneighboring or juxtaposed cells within the same tissue. A cellproliferative disorder includes a precancer or a precancerous condition.A cell proliferative disorder includes cancer. Preferably, the methodsprovided herein are used to treat or alleviate a symptom of cancer. Theterm “cancer” includes solid tumors, as well as, hematologic tumorsand/or malignancies. A “precancer cell” or “precancerous cell” is a cellmanifesting a cell proliferative disorder that is a precancer or aprecancerous condition. A “cancer cell” or “cancerous cell” is a cellmanifesting a cell proliferative disorder that is a cancer. Anyreproducible means of measurement may be used to identify cancer cellsor precancerous cells. Cancer cells or precancerous cells can beidentified by histological typing or grading of a tissue sample (e.g., abiopsy sample). Cancer cells or precancerous cells can be identifiedthrough the use of appropriate molecular markers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, uringarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi's sarcoma, kidney cancer, renal cancer, laryngealcancer, acute lymphoblastic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia,lip and oral cavity cancer, liver cancer, lung cancer, non-small celllung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstrammacroglobulinemia, medulloblastoma, melanoma, intraocular (eye)melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma,metastatic squamous neck cancer, mouth cancer, cancer of the tongue,multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, uterine cancer, uterinesarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cellskin carcinoma, small intestine cancer, soft tissue sarcoma, squamouscell carcinoma, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymoma,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter and other urinary organs, gestationaltrophoblastic tumor, urethral cancer, endometrial uterine cancer,uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer,and Wilm's Tumor.

A “lung cancer” is a cell proliferative disorder involving cells of thelung. In one aspect, lung cancer include all forms of cell proliferativedisorders affecting lung cells. In one aspect, lung cancer include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. In a preferred aspect, the method of the present disclosuremay be used to treat lung cancer or cell proliferative disorders of thelung. In one aspect, lung cancer includes all forms of cancer of thelung. In another aspect, lung cancer includes malignant lung neoplasms,carcinoma in situ, typical carcinoid tumors, and atypical carcinoidtumors. In another aspect, lung cancer includes small cell lung cancer(“SCLC”), non-small cell lung cancer (“NSCLC”), squamous cell carcinoma,adenocarcinoma, small cell carcinoma, large cell carcinoma,adenosquamous cell carcinoma, and mesothelioma. In another aspect, lungcancer includes “scar carcinoma,” bronchioalveolar carcinoma, giant cellcarcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. In one aspect lung cancer includes stage 0, IA, IB, IIA, IIB,IIIA, IIIB and IV lung cancer. In another aspect, lung cancer includeslung neoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

In one aspect, lung cancer include all forms of cell proliferativedisorders affecting lung cells. In one aspect, cell proliferativedisorders of the lung include lung cancer, precancerous conditions ofthe lung. In one aspect, cell proliferative disorders of the lunginclude hyperplasia, metaplasia, and dysplasia of the lung. In anotheraspect, lung cancer include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. In anotheraspect, cell proliferative disorders of the lung include replacement ofcolumnar epithelium with stratified squamous epithelium, and mucosaldysplasia. In another aspect, individuals exposed to inhaled injuriousenvironmental agents such as cigarette smoke and asbestos may be atincreased risk for developing cell proliferative disorders of the lung.In another aspect, prior lung diseases that may predispose individualsto development of cell proliferative disorders of the lung includechronic interstitial lung disease, necrotizing pulmonary disease,scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonitis,tuberculosis, repeated pneumonias, idiopathic pulmonary fibrosis,granulomata, asbestosis, fibrosing alveolitis, and Hodgkin's disease.

A “colon cancer” is a cell proliferative disorder involving cells of thecolon. In a preferred aspect, the method of the present disclosure maybe used to treat colon cancer or cell proliferative disorders of thecolon. In one aspect, colon cancer includes all forms of cancer of thecolon. In another aspect, colon cancer includes sporadic and hereditarycolon cancers. In another aspect, colon cancer includes malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. In another aspect, colon cancer includesadenocarcinoma, squamous cell carcinoma, and adenosquamous cellcarcinoma. In another aspect, colon cancer is associated with ahereditary syndrome selected from the group consisting of hereditarynonpolyposis colorectal cancer, familial adenomatous polyposis,Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome andjuvenile polyposis. In another aspect, colon cancer is caused by ahereditary syndrome selected from the group consisting of hereditarynonpolyposis colorectal cancer, familial adenomatous polyposis,Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome andjuvenile polyposis.

In one aspect, colon cancer include all forms of cell proliferativedisorders affecting colon cells. In one aspect, colon cancer includecolon cancer, precancerous conditions of the colon, adenomatous polypsof the colon and metachronous lesions of the colon. In one aspect coloncancer includes stage 0, I, IIA, IIB, IIC, IIIA, IIIB, IIIC, IVA, IVBand IVC colon cancer. In one aspect, a colon cancer includes adenoma. Inone aspect, colon cancer is characterized by hyperplasia, metaplasia ordysplasia of the colon. In another aspect, prior colon diseases that maypredispose individuals to development of cell proliferative disorders ofthe colon include prior colon cancer. In another aspect, current diseasethat may predispose individuals to development of cell proliferativedisorders of the colon include Crohn's disease and ulcerative colitis.In one aspect, a cell proliferative disorder of the colon is associatedwith a mutation in a gene selected from the group consisting of p53,ras, FAP and DCC. In another aspect, an individual has an elevated riskof developing a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “breast cancer” is a cell proliferative disorder involving cells ofthe breast. In a preferred aspect, breast cancer include all forms ofcell proliferative disorders affecting breast cells. In one aspect,breast cancer include breast cancer, a precancer or precancerouscondition of the breast, benign growths or lesions of the breast, andmalignant growths or lesions of the breast, and metastatic lesions intissue and organs in the body other than the breast. In another aspect,breast cancer include hyperplasia, metaplasia, and dysplasia of thebreast.

In one aspect, breast cancer is a precancerous condition of the breast.In one aspect, the method of the present disclosure may be used to treata precancerous condition of the breast. In one aspect, a precancerouscondition of the breast includes atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). In another aspect, a precancerous condition ofthe breast has been staged according to the TNM classification scheme asaccepted by the American Joint Committee on Cancer (AJCC), where theprimary tumor (T) has been assigned a stage of T0 or Tis; and where theregional lymph nodes (N) have been assigned a stage of N0; and wheredistant metastasis (M) has been assigned a stage of M0.

In one aspect, the method of the present disclosure may be used to treatbreast cancer. In one aspect, breast cancer includes all forms of cancerof the breast. In one aspect, breast cancer includes primary epithelialbreast cancers. In another aspect, breast cancer includes cancers inwhich the breast is involved by other tumors such as lymphoma, sarcomaor melanoma. In another aspect, breast cancer includes carcinoma of thebreast, ductal carcinoma of the breast, lobular carcinoma of the breast,undifferentiated carcinoma of the breast, cystosarcoma phyllodes of thebreast, angiosarcoma of the breast, and primary lymphoma of the breast.In one aspect, breast cancer includes Stage I, II, IIIA, IIIB, IIIC andIV breast cancer. In one aspect, ductal carcinoma of the breast includesinvasive carcinoma, invasive carcinoma in situ with predominantintraductal component, inflammatory breast cancer, and a ductalcarcinoma of the breast with a histologic type selected from the groupconsisting of comedo, mucinous (colloid), medullary, medullary withlymphcytic infiltrate, papillary, scirrhous, and tubular. In one aspect,lobular carcinoma of the breast includes invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. In one aspect, breast cancer includesPaget's disease, Paget's disease with intraductal carcinoma, and Paget'sdisease with invasive ductal carcinoma. In another aspect, breast cancerincludes breast neoplasms having histologic and ultrastructualheterogeneity (e.g., mixed cell types).

In one aspect, treating cancer results in a reduction in size of atumor. A reduction in size of a tumor may also be referred to as “tumorregression.” Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. In a preferred aspect, size of a tumor may be measured as adiameter of the tumor.

In another aspect, treating cancer results in a reduction in tumorvolume. Preferably, after treatment, tumor volume is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorvolume is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Tumor volume may be measured by any reproducible means of measurement.

In another aspect, treating cancer results in a decrease in number oftumors. Preferably, after treatment, tumor number is reduced by 5% orgreater relative to number prior to treatment; more preferably, tumornumber is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75%. Number oftumors may be measured by any reproducible means of measurement. In apreferred aspect, number of tumors may be measured by counting tumorsvisible to the naked eye or at a specified magnification. In a preferredaspect, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

In another aspect, treating cancer results in a decrease in number ofmetastatic lesions in other tissues or organs distant from the primarytumor site. Preferably, after treatment, the number of metastaticlesions is reduced by 5% or greater relative to number prior totreatment; more preferably, the number of metastatic lesions is reducedby 10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. The number of metastaticlesions may be measured by any reproducible means of measurement. In apreferred aspect, the number of metastatic lesions may be measured bycounting metastatic lesions visible to the naked eye or at a specifiedmagnification. In a preferred aspect, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

In another aspect, treating cancer results in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving carrier alone. Preferably, the average survivaltime is increased by more than 30 days; more preferably, by more than 60days; more preferably, by more than 90 days; and most preferably, bymore than 120 days. An increase in average survival time of a populationmay be measured by any reproducible means. In a preferred aspect, anincrease in average survival time of a population may be measured, forexample, by calculating for a population the average length of survivalfollowing initiation of treatment with an active compound. In anotherpreferred aspect, an increase in average survival time of a populationmay also be measured, for example, by calculating for a population theaverage length of survival following completion of a first round oftreatment with an active compound.

In another aspect, treating cancer results in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation of untreated subjects. Preferably, the average survival timeis increased by more than 30 days; more preferably, by more than 60days; more preferably, by more than 90 days; and most preferably, bymore than 120 days. An increase in average survival time of a populationmay be measured by any reproducible means. In a preferred aspect, anincrease in average survival time of a population may be measured, forexample, by calculating for a population the average length of survivalfollowing initiation of treatment with an active compound. In anotherpreferred aspect, an increase in average survival time of a populationmay also be measured, for example, by calculating for a population theaverage length of survival following completion of a first round oftreatment with an active compound.

In another aspect, treating cancer results in increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving a therapy that is not a recombinant polypeptide ofthe present disclosure. Preferably, the average survival time isincreased by more than 30 days; more preferably, by more than 60 days;more preferably, by more than 90 days; and most preferably, by more than120 days. An increase in average survival time of a population may bemeasured by any reproducible means. In a preferred aspect, an increasein average survival time of a population may be measured, for example,by calculating for a population the average length of survival followinginitiation of treatment with an active compound. In another preferredaspect, an increase in average survival time of a population may also bemeasured, for example, by calculating for a population the averagelength of survival following completion of a first round of treatmentwith an active compound.

In another aspect, treating cancer results in a decrease in themortality rate of a population of treated subjects in comparison to apopulation receiving carrier alone. In another aspect, treating cancerresults in a decrease in the mortality rate of a population of treatedsubjects in comparison to an untreated population. In a further aspect,treating cancer results in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a recombinant polypeptide of thepresent disclosure. Preferably, the mortality rate is decreased by morethan 2%; more preferably, by more than 5%; more preferably, by more than10%; and most preferably, by more than 25%. In a preferred aspect, adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. In another preferred aspect, adecrease in the mortality rate of a population may be measured, forexample, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith an active compound. In another preferred aspect, a decrease in themortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

In another aspect, treating cancer results in a decrease in tumor growthrate. Preferably, after treatment, tumor growth rate is reduced by atleast 5% relative to number prior to treatment; more preferably, tumorgrowth rate is reduced by at least 10%; more preferably, reduced by atleast 20%; more preferably, reduced by at least 30%; more preferably,reduced by at least 40%; more preferably, reduced by at least 50%; evenmore preferably, reduced by at least 50%; and most preferably, reducedby at least 75%. Tumor growth rate may be measured by any reproduciblemeans of measurement. In a preferred aspect, tumor growth rate ismeasured according to a change in tumor diameter per unit time.

In another aspect, treating cancer results in a decrease in tumorregrowth. Preferably, after treatment, tumor regrowth is less than 5%;more preferably, tumor regrowth is less than 10%; more preferably, lessthan 20%; more preferably, less than 30%; more preferably, less than40%; more preferably, less than 50%; even more preferably, less than50%; and most preferably, less than 75%. Tumor regrowth may be measuredby any reproducible means of measurement. In a preferred aspect, tumorregrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. In another preferred aspect, a decrease in tumor regrowth isindicated by failure of tumors to reoccur after treatment has stopped.

In another aspect, treating, preventing, or alleviating a cancer resultsin a reduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. In a preferredaspect, the rate of cellular proliferation is measured, for example, bymeasuring the number of dividing cells in a tissue sample per unit time.

In another aspect, treating, preventing, or alleviating a cancer resultsin a reduction in the proportion of proliferating cells. Preferably,after treatment, the proportion of proliferating cells is reduced by atleast 5%; more preferably, by at least 10%; more preferably, by at least20%; more preferably, by at least 30%; more preferably, by at least 40%;more preferably, by at least 50%; even more preferably, by at least 50%;and most preferably, by at least 75%. The proportion of proliferatingcells may be measured by any reproducible means of measurement. In apreferred aspect, the proportion of proliferating cells is measured, forexample, by quantifying the number of dividing cells relative to thenumber of nondividing cells in a tissue sample. In another preferredaspect, the proportion of proliferating cells is equivalent to themitotic index.

In another aspect, treating, preventing, or alleviating a cancer resultsin a decrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.In a preferred aspect, size of an area or zone of cellular proliferationmay be measured as a diameter or width of an area or zone of cellularproliferation.

In another aspect, treating, preventing, or alleviating a cancer resultsin a decrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. In one aspect, an abnormal cellular morphology ismeasured by microscopy, e.g., using an inverted tissue culturemicroscope. In one aspect, an abnormal cellular morphology takes theform of nuclear pleiomorphism.

In one aspect, treating cancer or a cell proliferative disorder resultsin cell death, and preferably, cell death results in a decrease of atleast 10% in number of cells in a population. More preferably, celldeath means a decrease of at least 20%; more preferably, a decrease ofat least 30%; more preferably, a decrease of at least 40%; morepreferably, a decrease of at least 50%; most preferably, a decrease ofat least 75%. Number of cells in a population may be measured by anyreproducible means. In one aspect, number of cells in a population ismeasured by fluorescence activated cell sorting (FACS). In anotheraspect, number of cells in a population is measured byimmunofluorescence microscopy. In another aspect, number of cells in apopulation is measured by light microscopy. In another aspect, methodsof measuring cell death are as shown in Li et al., (2003) Proc Natl AcadSci USA. 100(5): 2674-8. In a preferred aspect, cell death occurs byimmunogenic cell death.

Any of the above aspects can be combined with any other aspect asdisclosed herein.

Example 1: Methods of Producing Recombinant Polypeptides

Materials and Methods

The methods of producing the recombinant polypeptides of the presentdisclosure utilized the PCR primers disclosed in Table 3.

TABLE 3 Primer Sequences Primer Nucleotide Sequence (5′ to 3′)SEQ ID NO: A1 GGGGGGCATATGGACATTACCATCCAGCACCCCTGGTTCAAGCGCGCTC 33 T A2GGGGGGAAGCTTTTACTCCTCAGGCGCCTCGGTGGGCTT 34 ioE1CCTCTGTTCGAGGAGACTATCGAGCCCTACTA 35 ioE2TAGTAGGGCTCGATAGTCTCCTCGAACAGAGG 36 ioE3ACCGGCAGGAGCTGTTCCGCGAGGTGCTGTCGGAGGGCATTGAGTCGG 37 TGAGGGAGGACCGGGAioE4 TCCCGGTCCTCCCTCACCGACTCAATGCCCTCCGACAGCACCTCGCGGA 38ACAGCTCCTGCCGGT ioE5 ACTATGCTGGACGTAAAACACTTTGAGCCTTCGGACCTGGAGGTGAAG 39ATTA ioE6 TAATCTTCACCTCCAGGTCCGAAGGCTCAAAGTGTTTTACGTCCAGCAT 40 GAT ioE7AAGATTATCGACGACTTTGTGTCGATCCATGGC 41 ioE8GCCATGGATCGACACAAAGTCGTCGATAATCTT 42 ioE9GGCAAGCACGAGTCGAGACAGGACGACCACGGCTACATCGAGCGGTC 43 GTTTCACCGC ioE10GCGGTGAAACGACCGCTCGATGTAGCCGTGGTCGTCCTGTCTCGACTCG 44 TGCTTGCC ioE11GCGGACCAGGAGGCCATCACCTGCGAGCTGGAGGGCGACGG 45 ioE12CCGTCGCCCTCCAGCTCGCAGGTGATGGCCTCCTGGTCCAC 46 ioE13TTCGACCAGTTTTTCGGATCGGGTCTGCTGTCGTATGACCTGCTGCCTCT 47 GTTC ioE14GGGGACCTTGGGGCCCTCGAAGGTCAGCATGCCGTCGCC 48 ioE15TTCAAGCGCGCTCTGGGACCCCTGATTCCAGAGCGTCTGTTCGACCAGT 49 TTTTCGGA ioE16CACGGGGATGGGCCTCGACTCGTGGGTGGGGTCCATGTTCTCGGGGAC 50 CTTGGGG ioE17ATGGACATTACCATCCAG 51 ioE18AAGCTTTTACTCCTCAGGCGCCTCGGTGGGCTTCGACGACCGCTCCACG 52 GGGATGGGCCT

Preparation of Template DNA

The full length CRYAA sequence from Anser cygnoides domesticus (SEQ IDNO: 17) was amplified in a PCR reaction using Pfu polymerase. A1 primer(SEQ ID NO: 33) and A2 primer (SEQ ID NO: 34) were used in the PCRreaction. The gene was cloned into NdeI and HindIII sites in a pET24avector (Novagen) using the manufacturer's protocol. The ligation mixturewas transformed into Escherichia coli DH5alpha cells and transformantswere selected on LB ampicillin plates. Plasmid DNA was isolated fromseveral transformants and screened by restriction digestion of NdeI andHindIII sites. A sequence verified clone containing Anser cygnoidesdomesticus CRYAA (SEQ ID NO: 17) was identified and used as template.

Cloning of Plasmid Containing the CRYA_1B Recombinant PolypeptideSequence

The recombinant plasmid containing CRYA_1B (SEQ ID NO: 25) was preparedin the following manner. PCR was performed using the template DNAdescribed above, forward primer IoE1 (SEQ ID NO: 35) and reverse primerIoE2 (SEQ ID NO: 36). PCR temperature and time were programmed asfollows: denaturing at 95° C. for 5 minutes; followed by 30 cycles ofPCR reactions with denaturation at 95° C. for 30 sec, annealing at 60°C. for 30 sec, and elongation at 72° C. for 1 minute; final elongationat 72° C. for 10 minutes. All PCR amplifications were performed with PfuUltra polymerase (Stratagene). PCR products were separatedelectrophoretically using 1.0% agarose gel, and stained with ethidiumbromide. The DNA fragment was extracted from the gel using GFX™ PCR DNAand Gel Bind Purification Kit (GE Healthcare) and ligated into a pET24a(Novagen) vector. The ligation mixture was transformed into the DH5alphaEscherichia coli strain and transformants were selected on LB platescontaining ampicillin. Plasmid DNA was isolated from transformants. Asequence verified clone, Plasmid_1, was used as a template for asubsequent round of PCR amplification.

PCR amplification was performed using Plasmid_1, forward primer IoE3(SEQ ID NO: 37) and reverse primer IoE4 (SEQ ID NO: 38). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, 32 cyclesof (95° C. for 30 seconds, 65° C. for 30 seconds, 72° C. for 1 minute),followed by 5 minutes at 72° C. The PCR product was purified and clonedinto a pET24a plasmid using NdeI and HindIII restriction sites. Asequence verified clone, Plasmid_2, was used as a template for asubsequent round of PCR amplification.

PCR amplification was performed using Plasmid_2, forward primer IoE5(SEQ ID NO: 39) and reverse primer IoE6 (SEQ ID NO: 40). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 58° C. for 30 seconds, 72° C. for 1 minute in35 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_3,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_3, forward primer IoE7(SEQ ID NO: 41) and reverse primer IoE8 (SEQ ID NO: 42). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 55° C. for 30 seconds, 72° C. for 1 minute in28 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_4,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_4, forward primer IoE9(SEQ ID NO: 43) and reverse primer IoE10 (SEQ ID NO: 44). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 53° C. for 30 seconds, 72° C. for 1 minute in33 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_5,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_5, forward primer IoE11(SEQ ID NO: 45) and reverse primer IoE12 (SEQ ID NO: 46). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 57° C. for 30 seconds, 72° C. for 1 minute in30 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_6,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_6, forward primer IoE13(SEQ ID NO: 47) and reverse primer IoE14 (SEQ ID NO: 48). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 51° C. for 30 seconds, 72° C. for 1 minute in32 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_7,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_7, forward primer IoE15(SEQ ID NO: 49) and reverse primer IoE16 (SEQ ID NO: 50). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 54° C. for 30 seconds, 72° C. for 1 minute in32 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. A sequence verified clone, Plasmid_8,was used as a template for a subsequent round of PCR amplification.

PCR amplification was performed using Plasmid_8, forward primer IoE17(SEQ ID NO: 51) and reverse primer IoE18 (SEQ ID NO: 52). PCRamplification and cloning were performed using the procedure describedabove and the following PCR conditions: 95° C. for 5 minutes, followedby 95° C. for 30 seconds, 52° C. for 30 seconds, 72° C. for 1 minute in32 cycles, with a final 5 minute extension at 72° C. PCR products wereseparated electrophoretically using 1.0% agarose gel, and stained withethidium bromide. The DNA fragment was excised from the gel, extractedand cloned into a pET24a plasmid. The ligation mixture was transformedinto DH5alpha strain of Escherichia coli cells and transformants wereselected on LB plates containing ampicillin. A sequence verified clone,Plasmid_9 contains the CRYA_1B (SEQ ID NO: 25) in the correct readingframe.

Expression of Recombinant Polypeptide CRYA_1B

Plasmid_9 was transformed into the expression Escherichia coli strainBL21, and the ampicillin-resistant colonies were selected. The expectedmolecular weight for CRYA_1B recombinant polypeptide was 20 kDa (FIG.2). A single colony from Luria-Betani (LB)-agar plate supplemented with100 μg/ml ampicillin was selected. In this preparation, a 50 ml conicaltube containing 3 ml of LB medium (10 g tryptone, 10 g NaCl and 5 gyeast extract per L) and 100 μg/ml of ampicillin was inoculated with asingle colony and grown overnight in a shaking incubator set at 37° C.and 200 RPM. The culture was further expanded by adding 3 ml of theculture into a sterile 500 ml Erlenmeyer flask containing 100 ml of 2YTmedium (16 g tryptone, 15 g yeast extract and 8 g NaCl per L) and 100μg/ml of ampicillin and grown overnight in a shaking incubator set at37° C. and 200 RPM. This resulted in a seed culture.

A 6 L bioreactor was used to further expand the seed culture. 4 L of 2YTmedium containing 100 μg/ml of ampicillin was inoculated with 100 ml ofseed culture grown overnight in a shaking incubator set at 37° C. and200 RPM. In the bioreactor, cultures were incubated at 37° C., airflowand agitation of 2 SLPM (standard liners per minute) and 200 RPM. Whenthe OD600 reached 0.65 to 0.75, protein overexpression was induced with1.0 mM Isoropyl-β-D-thiogalactopyranoside (IPTG). The cells were allowedto grow for 7 to 8 hours and the agitation speed, temperature and airflow were set to 400 RPM, 28° C. and 4 SLPM, respectively. To controlfoaming, Polyglycol P-2000 antifoam was added as required. After 7 to 8hours of induction, the cells were harvested by centrifugation at 8000rpm for 15 minutes at 4° C. The cell pellets were frozen and stored at−80° C.

Purification of Recombinant Polypeptide CRYA_1B

In this preparation, the pellets, equivalent to 6 g of CRYA_1Brecombinant polypeptide (SEQ ID NO: 9) was resuspended in 40 ml ofBuffer A (50 mM Tris-HCl buffer) and disrupted by sonication on ice (28cycles of 10-s pulses with 30-s intervals, 30% amplitude using anultrasonic cell disruptor Misonix Ultrasonic Liquid Processors S-4000,USA) to obtain the total protein extract for solubility analysis. Thetotal protein extract was centrifuged at 14,000 rpm for 45 min at 4° C.using a Sorvall RCSC Plus (USA) ultracentrifuge using a type SS-34rotor. The supernatant was filtered through a 0.45 μm filter (Millipore)and loaded onto a Q-Sepharose anion exchange column equilibrated in thesame buffer. Q-Sepharose was packed into a C 26/40 Column (GEHealthcare) to a bed height of 20 cm. A 40 mL volume of supernatantcontaining CRYA_1B recombinant polypeptide (SEQ ID NO: 9) was loadedonto the column using AKTA FPLC (GE Healthcare) at a flow rate of 5ml/min. CRYA_1B recombinant polypeptide (SEQ ID NO: 9) was eluted by aconcentration gradient by using an equilibrium buffer containing 50 mMTris-HCl, NaCl buffer and collected in a single peak based on A280absorbance for further application on the hydrophobic interactioncolumn. The eluents collected were analyzed by 15% SDS-polyacrylamidegel electrophoresis.

Hydrophobic Interaction Chromatography

After ion exchange chromatography, the eluted CRYA_1B recombinantpolypeptide (SEQ ID NO: 9) was pooled together, concentrated by AmiconUltra 15 ml Centrifugal Filter (Merck), and subsequently added to thesaturated ammonium sulphate buffer (50 mM Tris-HCl, 3.8 M ammoniumsulphate, 1 mM DTT and 1 mM EDTA), resulting in a final concentration of1.2 M ammonium sulphate. The concentrated product with adding ammoniumsulphate was filtered using a 0.45 μm syringe filter (Millipore) andloaded to Hydrophobic Interaction Chromatography column (C 10/20 column,Ge Healthcare) at a flow rate of 2 ml/min. Source 15PHE (GE Healthcare)was packed into C 10/20 column (GE Healthcare) to a bed height of 10 cmand pre-equilibrated with buffer A (50 mM Tris-HCl, 1.2 M ammoniumsulphate, 10% glycerol, 1 mM DTT and 1 mM EDTA). The column was washedwith buffer A and the protein elution using buffer B (50 mM Tris-HCl,10% glycerol, 1 mM DTT and 1 mM EDTA) was achieved with a lineargradient with decreasing ammonium sulphate and increasing glycerol. Theeluted protein was analyzed by 15% SDS-PAGE. The fractions were furtherconcentrated by Amicon Ultra 15 ml Centrifugal Filter (Merck).

Buffer Exchange Using Gel Filtration

The purified recombinant polypeptide was further exchanged into PBSbuffer by using a Sephadex G-25 column. Sephadex G-25 was packed into aC 26/100 column (GE Healthcare) to a bed height of 85 cm andpre-equilibrated with PBS buffer at a flow rate of 1 ml/min. Theconcentrated protein was eluted after 2.5 hours and analyzed by 15%SDS-PAGE. The resulting eluates were then concentrated by Amicon Ultra15 ml Centrifugal Filter (Merck).

Example 2: Methods of Inducing or Enhancing an Immune Response

Cancer cell lines were treated with CRYA_1B recombinant polypeptide (SEQID NO: 9), CRYA_1B recombinant polypeptide was diluted to variousconcentrations and incubated with human cancer cell lines H441 (lungcancer, HTB-174, ATCC), H460 (lung cancer, HTB-177, ATCC), HCT15 (coloncancer, CCL-225, ATCC) and MCF7 (breast cancer, HTB-22, ATCC) all at 37°C. As of CRT, HSP70, HSP90, and Caspase 3/7 assay, the recombinantpolypeptide incubation time for H441 is 60 min, 1 hr50 min, 1 hr40 min,and 2 hr45 min respectively, for H460 is 30 min, 1 hr15 min, 1 hr5 min,and 2 hr30 min respectively, for HCT15 is 55 min, 1 hr50 min, 1 hr30min, and 2 hr30 min respectively, and for MCF7 is 1 hr10 min, 1 hr40min, 1 hr45 min, and 2 hr45 min respectively. Flow cytometry was used toassess the cell surface expression of calreticulin (CRT) (FIGS. 3-6),HSP70 (FIGS. 7-10), HSP90 (FIGS. 11-14) and Caspase 3/7 (FIGS. 15-18) incells treated with CRYA_1B recombinant polypeptide and in untreatedcontrol cells. This was performed using a FACSCalibur (BD Biosciences)using CRT mAb (Abcam), HSP70 mAb (Enzo Life Sciences), HSP90 mAb (EnzoLife Sciences) and Caspase 3/7 (Invitrogen assay), respectively.

What is claimed is:
 1. A recombinant polypeptide comprising an amino acid sequence of SEQ ID NO:
 9. 2. A pharmaceutical composition comprising the recombinant polypeptide of claim 1 and a pharmaceutically acceptable carrier.
 3. The pharmaceutical composition of claim 2, wherein the final NaCl concentration is 0.4M-1.0M and the pharmaceutically acceptable carrier comprises a buffer solution of pH 7.5-9.0. 